Resumo. Abstract Aims: to evaluate the richness, biomass and distribution of aquatic macrophytes in a subtropical reservoir in the dry and rainy seasons. Methods this study was carried out in the Itupararanga Reservoir, an important water source in São Paulo State, undergoing a continuous process of eutrophication. Samples of macrophytes were collected at 12 sampling sites in the summer and at 9 sampling sites in the winter in the Itupararanga Reservoir using the quadrat method (0.25 m2). In the laboratory, the plants were washed to remove the coarse material and then were dried (60 °C) for biomass determination (gDW. m-2). All the species in the sampling sites in both periods were identified using the specific literature. In each sampling site, the water temperature, pH, electrical conductivity and dissolved oxygen were measured using a probe. The temporal and spatial differences were analyzed using t-test and a Cluster Analysis was performed. Results The checklist showed sixteen species, 75% of them were emergent. From the 16 species, 15 were present in the summer and 10 in the winter. Eichhornia crassipes, Polygonum sp., and Urochloa sp. were the frequent taxa and had the highest biomass in both periods. The winter showed the highest biomass mainly due to the growth of free-floating species. The headwaters of the reservoir, the most eutrophic region, showed that the highest macrophyte richness and the sampling sites of this area were clustered in both the summer and winter. Conclusions There was no significant spatial variation among the measured variables. E. crassipes, Salvinia sp. and Urochloa sp. showed a significant variation of biomass between two periods. Urochloa sp. is a nuisance species occurring in up to 60% of the sampling sites having implications for the whole catchment. Continuous macrophyte monitoring is important due to the increasing trophic status of this ecosystem.