Martim Afonso de Sousa (c. 1500 – 21 July 1564) was a Portuguese fidalgo, explorer and colonial administrator.

Martim Afonso de Sousa was born in Vila Viçosa, and had been raised in the Duke of Bragança household and was a cousin and personal friend since childhood of King John III of Portugal.

When he left the service of the Duke of Bragança, in 1516, to stay at the court of the Royal House, he began to study mathematics, cosmography and geography with the chief cosmographer Pedro Nunes. He justified the decision by saying: "The duke can make me mayor, but the king can make me duke", but that never happened.

After the death of Manuel I of Portugal, he accompanied the widowed Queen D. Leonor of Austria to Castile, where he married Ana Pimentel, from an illustrious Spanish family, around June 1523. Ana Pimentel was the daughter of Arias Maldonado, commendator of Estriana and governor of Salamanca and Talavera and of D. Joana Pimentel, daughter of D. Pedro Pimentel, Lord of Távara and sister of D. Bernardino Pimentel, 1st Marquis of Távara. The mother of Martim Afonso de Sousa's wife was thus a descendant, on her paternal side, of the Pimentel Counts of Benavente, a lineage of Portuguese origin that passed to Spain in the 14th century; and, on her maternal side, she descended from the Counts of Alba de Liste. This means that, through his marriage, Martim Afonso de Sousa became linked to some of the main lineages in Spain. At the time, it was unusual for non-titled Portuguese noblemen to marry Spanish women who came — even if, as in this case, through female origin — from families of the greatness of Spain, so it can be concluded that Martim Afonso managed to successfully develop a strategy of marriage alliance with positive repercussions in terms of his political career and connections with power in the first court of the Iberian Peninsula.

In Spain, where he lived for four years, he fought in the service of Emperor Charles V against the French. In the winter of 1525, Martim Afonso played a prominent role in the siege and capture of Fuenterrabía, in northwestern Spain. The emperor publicly praised him and invited him to remain in Castile.

Around 1530, King John III determined to send an official Portuguese expedition to Brazil, and Martim Afonso de Souza was appointed by his first cousin Dom António de Ataíde, 1st Count of Castanheira, member of the Royal Council. Ataíde made this appointment with the intention of distancing Martim Afonso from King John III, so that he would be the only one to have a certain influence on the King.

Threatened by the presence of French ships along the coast of Brazil, the Portuguese crown in December 1530 sent a fleet with 400 people led by Martim Afonso de Sousa to establish control and explore. His mission was to place Portuguese markers as far south as the River Plate estuary, but he was shipwrecked there.

Upon his return to São Vicente and Santos, in 1532 he led troops guided by the native inhabitants and by earlier Portuguese settlers such as João Ramalho up the Serra do Mar mountains to the area near the future village of São Paulo. On the high plateau, he founded the town of Santo André. He also established a sugar mill near the coast at São Vicente, with sugarcane brought from the Portuguese Cape Verde islands. In both activities, Afonso de Sousa established a pattern followed by Portuguese colonizers and Brazilians for long afterward: the "entradas" and "bandeiras" – or explorations and raids into the interior – and the production of sugar along the coast for export.