Beta diversity is the variability in species composition among sampling units for a given area and can be influenced by several environmental drivers, including environmental heterogeneity. Here, we considered the contribution of seven tributaries flowing into the Upper Paraná River channel as the mains drivers of environmental heterogeneity and zooplankton beta diversity. We used Mantel test to examine the relationships between zooplankton functional beta diversity components (total, turnover, and nestedness) and environmental and geographical distance. Generalized dissimilarity modeling (GDM) was run to test which environmental variables were the best predictors of beta diversity components. Mantel’s test results revealed that total beta diversity was positively related with environmental heterogeneity in almost all periods. GDM analysis results showed that total beta and turnover were related to temperature, organic suspended matter, dissolved oxygen, NH4, and pH, while nestedness was influenced by depth and geographic distances. Our results support the idea that smaller rivers are a main source of diversity for large rivers, especially the ones with cascading reservoirs. Overall, our study shoes that variation in limnological variables results in higher dissimilarity in zooplankton communities and that environmental change filters and sorts species according to their functional traits.