Resumo. Abstract Aim To evaluate the effects of environmental heterogeneity promoted by thermal stratification on the distribution of limnologic variables and phytoplankton functional groups (FGs) in two natural lakes. Methods Monthly measurements were performed over a five-year period in the vertical profile of a warm monomitic shallow lake (Lago Carioca) and in a deep and meromictic (Lake Dom Helvécio). Results The vertical zonation generated by the high thermal stability during the stratification period promoted an increase in the spatial heterogeneity and, consequently, in the richness of functional groups of the two lakes. In the epilimnion, the dominance of small chlorophytes and desmids (NA, A and X1, ≤20μm) and larger dinophyceans (Lo, ≥20μm) were associated with high turbulence and light availability and soluble phosphorus limitation. In the metalimnion, the presence of filamentous cyanobacteria (R) and colonial chlorophyceans (F) of larger size (≥20µm) were associated with stable habitats with high concentrations of N-NH4 and P-PO4-3 and light limiting conditions. Comparatively, Lake Dom Helvécio presented a higher richness of FGs in the meta-hipolimnetic layers (SN, P) as well as a higher number of species per functional group. Seasonal changes in the climatic conditions (e.g. the decrease in air temperature with the consequent heat loss) caused the break of the water column stability, which promoted the redistribution of the dissolved nutrient forms and the increase of light limitation in the two lakes during the mixing period. Therefore, there was a drastic reduction in the richness and population biomass of FGs (≤80%). Conclusions Thermal stability and atelomixis were the main driving forces of vertical heterogeneity during the stratification, favoring the coexistence of FGs and, consequently, their increase in richness and biomass.