The discontinuity in rivers caused by the construction of reservoirs disrupts the longitudinal gradient and decreases the natural connectivity of lotic systems. We evaluated the dynamics of Reynolds functional groups (RFGs) in response to the formation of a subtropical reservoir, in relation to the environmental changes between the pre-and post-impoundment periods. We hypothesized that the disruption of longitudinal connectivity and the changes in the light and mixing regime after the impoundment would directly influence the phytoplankton functional structure. Significant changes in the light and mixing regimes were observed after the impoundment, with the formation of a lacustrine zone, influencing the dynamics of RFGs. Fourteen RFGs were recorded. RFG C (diatoms) dominated in the pre-impoundment period. In the post-impoundment period, RFGs C, P (diatoms), and J (chlorophyceans) codominated with the highest mean values of biovolume. RFGs MP, N, H1, W2, F, E, A, W1, B, X1, and S1 contributed relatively little to the biovolume, although with higher values in the post-impoundment period. RFGs F and S1 were recorded only after the impoundment. This study revealed the important roles of physical processes and nutrients in the spatial and seasonal gradient, and in the selection of phytoplankton functional groups in a subtropical reservoir.