Lake stratification is the tendency of lakes to form separate and distinct thermal layers during warm weather. Typically stratified lakes show three distinct layers: the epilimnion, comprising the top warm layer; the thermocline (or metalimnion), the middle layer, whose depth may change throughout the day; and the colder hypolimnion, extending to the floor of the lake.

Every lake has a set mixing regime that is influenced by lake morphometry and environmental conditions. However, changes to human influences in the form of land use change, increases in temperature, and changes to weather patterns have been shown to alter the timing and intensity of stratification in lakes around the globe. Rising air temperatures have the same effect on lake bodies as a physical shift in geographic location, with tropical zones being particularly sensitive. These changes can further alter the fish, zooplankton, and phytoplankton community composition, in addition to creating gradients that alter the availability of dissolved oxygen and nutrients.

The thermal stratification of lakes refers to a change in the temperature at different depths in the lake, and is due to the density of water varying with temperature. Cold water is denser than warm water, and the epilimnion generally consists of water that is not as dense as the water in the hypolimnion. The temperature of maximum density for fresh water is 4 °C. In temperate regions where lake water warms up and cools through the seasons, a cyclical pattern of overturn occurs that is repeated from year to year as the cold dense water at the top of the lake sinks (see stable and unstable stratification). For example, in dimictic lakes the lake water turns over during the spring and the fall. This process occurs more slowly in deeper water; as a result, a thermal bar may form. If the stratification of water lasts for extended periods, the lake is meromictic.

Heat is transported very slowly between the mixed layers of a stratified lake: the diffusion of heat just one vertical meter takes about a month. The interaction between the atmosphere and lakes depends on how solar radiation is distributed, which is why water turbulence, mainly caused by wind stress, can greatly increase the efficiency of heat transfer. In shallow lakes, stratification into epilimnion, metalimnion, and hypolimnion often does not occur, as wind or cooling causes regular mixing throughout the year. These lakes are called polymictic. There is not a fixed depth that differentiates polymictic and stratifying lakes, as this is also influenced by turbidity, lake surface area, and climate.

The lake mixing regime (e.g. polymictic, dimictic, meromictic) describes the yearly patterns of lake stratification that occur in most years. However, short-term events can influence lake stratification as well. Heat waves can cause periods of stratification in otherwise mixed, shallow lakes, while mixing events, such as storms or large river discharge, can break down stratification. Weather conditions induce a more rapid response in larger, shallower lakes, so these lakes are more dynamic and less well understood. However, mixing regimes that are known to exist in large, shallow lakes are mostly diurnal, and the stratification is easily disturbed. Lake Taihu in China is an example of a large, shallow, diurnal lake, where even though the depth does not reach more than 3 meters (10 ft), the lake's water turbidity is still dynamic enough to stratify and de-stratify due to the absorption of solar radiation, mostly in the upper layer. The tendency for stratification to become disrupted affects the rate of transport and consumption of nutrients, in turn affecting the presence of algal growth. Stratification and mixing regimes in Earth's largest lakes are also poorly understood, yet changes in thermal distributions, such as the rising temperatures found over time in Lake Michigan's deep waters, can significantly alter the largest freshwater ecosystems on the planet.

Recent research suggests that seasonally ice-covered dimictic lakes may be described as "cryostratified" or "cryomictic" according to their wintertime stratification regimes. Cryostratified lakes exhibit inverse stratification near the ice surface, and have depth-averaged temperatures near 4 °C, while cryomictic lakes have no under-ice thermocline and have depth-averaged winter temperatures closer to 0 °C.

Water circulation during mixing periods causes the movement of oxygen and other dissolved nutrients, distributing them throughout the body of water. In lakes where benthic organisms are prominent, the respiration and consumption of these bottom-feeders may outweigh the mixing properties of strongly stratified lakes, resulting in zones of extremely low near-bottom oxygen and nutrient concentrations. This can be harmful to benthic organisms such as shellfish; in the worst cases it can wipe out entire populations. The accumulation of dissolved carbon dioxide (CO₂) in three meromictic lakes in Africa (Lake Nyos and Lake Monoun in Cameroon and Lake Kivu in Rwanda) is potentially dangerous, because if one of these lakes is triggered into limnic eruption, a very large quantity of CO₂ can quickly leave the lake and displace the oxygen needed for life by people and animals in the surrounding area.

In temperate latitudes, many lakes that become stratified during the summer months de-stratify during cooler windier weather, with surface mixing by wind being a significant driver in this process. This is often referred to as "autumn turn-over". The mixing of the hypolimnium into the mixed water body of the lake recirculates nutrients, particularly phosphorus compounds, trapped in the hypolimnion during the warm weather. It also poses a risk of oxygen sag as a long established hypolimnion can be anoxic or very low in oxygen.