The performance of individual phytoplankton species is strongly governed by the thermal stratification's impact on vertical mixing within the water column, which alters the position of phytoplankton relative to nutrients and light. The present study documents shifts in phytoplankton structure and vertical positioning that have accompanied intensified long-term stratification in a natural ecosystem. Ordination analysis is used to extract gradients in phytoplankton composition in Lake Tahoe, an extremely nutrient-poor lake, over a 23-year period of records. Community structure in the 1980s was associated most strongly with resource availability (low nitrogen to phosphorus ratios, deeper euphotic zone depth), while intensified stratification dominated the phytoplankton structure since the late 1990s. Within diatoms, small-sized cells increased with reduced mixing, suggesting that suppressed turbulence provides them with a competitive advantage over large-sized cells. Among the morphologically diverse chlorophytes, filamentous and coenobial forms were favored under intensified stratification. The selection for small-sized diatoms is accompanied by a shoaling trend in their vertical position in the water column. In contrast, the motile flagellates displayed a deeper vertical positioning in recent years, indicating that optimal growth conditions shifted likely due to reduced upwelling of nutrients. As the thermal stratification of lakes and oceans is strongly linked to climate variables, the present study confirms that climate warming will alter phytoplankton structure and dynamics largely through effects on nutrient availability and sinking velocities. Intensified stratification should favor the expansion of small-sized species and species with the capability of buoyancy regulation, which may alter primary productivity, nutrient recycling, and higher trophic productivity.