The Mytilenean revolt was an incident in the Peloponnesian War in which the city of Mytilene attempted to unify the island of Lesbos under its control and revolt from the Athenian Empire. In 428 BC, the Mytilenean government planned a rebellion in concert with Sparta, Boeotia, and certain other cities on the island, and began preparing to revolt by fortifying the city and laying in supplies for a prolonged war. These preparations were interrupted by the Athenian fleet, which had been notified of the plot, and the Mytileneans sent representatives to Athens to discuss a settlement, but simultaneously dispatched a secret embassy to Sparta to request support.

The attempt to reach a settlement at Athens fell through, as the Athenians were unwilling to allow their loyal ally Methymna to be subjugated by the Mytileneans, and the Athenian fleet blockaded Mytilene by sea. Sparta, although it agreed to send support and prepared a fleet, was cowed by an Athenian show of force and took no action at this time. On Lesbos, meanwhile, the arrival of 1,000 Athenian hoplites allowed Athens to complete the investment of Mytilene by walling it in on land. Although Sparta finally dispatched a fleet in the summer of 427 BC, it advanced with such caution and so many delays that it arrived in the vicinity of Lesbos only in time to receive news of Mytilene's surrender.

In the wake of the Mytileneans' surrender, a heated debate took place at Athens over their fate. One faction, led by Cleon, advocated executing all of the men in the city and enslaving the women and children, while another faction (one spokesman was Diodotus) preferred more moderate treatment in which only men who had been identified as ringleaders would be executed. The Athenian assembly wavered; an order for mass execution was issued on the first day of debate but countermanded on the next. In the end, the city as a whole was spared, but 1,000 "ringleaders" (although this figure is viewed sceptically, and it is believed that due to a misreading by a scribe, the figure was actually closer to 30) were executed without trial.

The Mytilenean government (which was oligarchic) had considered revolting from Athens even before the Peloponnesian War broke out, but when they initially approached Sparta in the 430s BC, the Spartans would not promise to accept them into the Peloponnesian League. Without the necessary Spartan support that would have made revolt feasible, the Mytileneans' plan came to nothing. In 428, however, the Mytilenean leaders judged that the time was ripe for revolt, and both Boeotia and Sparta participated in planning the rebellion. The primary motivation for the rebellion was the Mytilenean's desire to gain control of all of Lesbos; Athens generally discouraged the creation of multi-city subunits of the empire, and would certainly not have permitted Lesbos to be unified. Moreover, Mytilene's privileged status as an independent state, commanding its own fleet, within the Athenian empire seems to have given its leaders both confidence in their chances of success and concern that, if they did not revolt, they might in the future be reduced to the same tributary status as the majority of Athens' allies. The Mytileneans, therefore, began strengthening their fortifications and sent for mercenaries and supplies from the Black Sea region. Before they had completed their preparations, however, their plans were betrayed to the Athenians by several of their enemies in the region, namely the Methymnians and Tenedians, and by a group of Mytilenean citizens who represented Athens' interests in that city (probably members of the democratic faction there).

The Athenians, who were still suffering from the plague at this time and were under great financial strain from the unexpectedly long and involved war, initially tried to negotiate in order to avoid taking on yet another military commitment in Lesbos. When the Mytileneans refused to abandon their plans to unify Lesbos or their preparations for war, however, the Athenians resigned themselves to the necessity of a military response and dispatched a fleet to Mytilene; ten Mytilenean triremes which had been serving in the fleet were interned at Athens with their crews. The initial plan was for the fleet to arrive during a religious festival for which all the Mytileneans would be outside of the city, and during which it would be easy for the Athenians to seize the fortifications of the town. Since this plan was crafted in the open Athenian assembly, however, it was impossible to keep it secret, and the Mytileneans had ample warning of the fleet's approach. On the day of the festival, they remained in the city, with doubled guards on the weaker sections of the walls; the Athenians, arriving to find the city well defended, ordered the Mytileneans to surrender their fleet and tear down their walls. The Mytileneans refused this demand, and even went so far as to send their fleet to fight the Athenians just outside the harbor. When the Athenians quickly defeated this fleet and drove it back into the harbor, however, the Mytileneans quickly agreed to negotiate, arranged an armistice on the scene, and sent representatives to Athens. In doing this, however, the Mytilenean government was aiming not at reaching an accommodation with Athens, but rather at buying time for their negotiations with Sparta and Boeotia to bear fruit. As the representatives were on their way to Athens, a second group was secretly dispatched to Sparta to secure that city's support in the rebellion.

The negotiations at Athens were brief and unsuccessful. The Mytileneans offered to remain loyal if the Athenians would withdraw their fleet from Lesbos. Implicit in this proposition was that the Athenians would abandon Methymna, and that the Athenians could not do, as failing to protect a subject city from aggression would have undermined their claims to legitimacy as rulers of their empire. The Athenians, accordingly, rejected the Mytilenean offer, and when the ambassadors returned to Lesbos bearing this news all the cities of Lesbos save Methymna openly declared war on Athens. The Mytileneans mustered an army and marched out to attack the Athenian camp; although they came off slightly the better in the ensuing battle, they were unwilling to press their advantage and retreated back behind their fortifications before nightfall. At this point the Athenians, encouraged by the lack of initiative on their enemies' part, summoned troops from their allies and, once these arrived, built two fortified camps, one on either side of Mytilene's harbor. From these they instituted a naval blockade of the city (the Mytileneans and their allies continued to control all the land outside the Athenian fortifications).

Immediately after the Mytilenean attack on the Athenian camp, a trireme bearing ambassadors from Sparta and Boeotia slipped past the Athenians into Mytilene and persuaded the Mytileneans to send a second group of ambassadors to plead for Spartan intervention (the Spartans and Boeotians had been dispatched before the revolt, but had been prevented from entering the city for some time). This second group of Mytilenean ambassadors arrived within a week of the first, in July, but neither secured any immediate assistance; the Spartans deferred the decision over Mytilene to the Peloponnesian League as a whole, which would be convening at Olympia later that summer. At that meeting, the Mytilenean ambassadors gave a speech in which they gave justifications for their revolt, emphasized Athens' weakness, and stressed the importance of attacking the Athenians in the empire, from which they drew their resources. After hearing this speech, the Spartans and their allies voted to accept the Lesbians into their alliance and attack Athens immediately in support of the revolt.

The plans made at Olympia called for all the allied states to send their contingents to the isthmus of Corinth to join together and prepare to advance on Athens. The Spartan contingent was the first to arrive, and set about dragging ships across the isthmus from the gulf of Corinth so as to be able to attack simultaneously on land and sea. While the Spartans set enthusiastically about this work, however, the other allies sent in their contingents only slowly; the harvest was underway, and the allies were tired of constant military service (their service had already been called on that summer for a month-long invasion of Attica beginning in May). The Athenians, meanwhile, aware that the Peloponnesian's readiness to attack derived in part from the Mytileneans' assertions that Athens was critically weakened, prepared a fleet of 100 ships to raid the coast of the Peloponnese. Preparing the fleet required extreme measures, as the state's resources were already stretched thin; as not enough thetes (poor citizens) were available for service to crew the fleet fully, both zeugitai (landowners who usually fought as hoplites) and metics (resident aliens) were recruited to serve as rowers. The fleet raided at will along the Peloponnesian coast, and the Spartans, who had been promised that the forty ships at Mytilene and the forty that had circumnavigated the Peloponnese earlier in the summer were all the Athenians could muster, concluded that they had been deceived and called off their plans to launch an attack that summer.