Beta diversity represents the spatial or temporal variation of species diversity among sampling sites and may be composed of two elements: turnover (Brepl, replacement of species assemblages) and nestedness (Brich, loss or gain of species assemblages). Knowledge of the mechanisms driving beta diversity contributes to an understanding of the variation in aquatic ecosystem community structures. We sampled zooplankton assemblages at 24 sites in 11 rivers in Shanghai City and conducted a nutrient addition experiment to elucidate the effects of various environmental variables on the community structure and beta diversity patterns of the zooplankton. The zooplankton assemblages in the rivers differed significantly at ammonia nitrogen (NH3-N) concentrations below (Group I) and above (Group II) 1.03 mg·L−1. The nutrient addition experiment further demonstrated that the composition of the zooplankton assemblages changed markedly along an ammonia concentration gradient (0.2 to 5.0 mg N·L−1). The total beta diversity of Group I was considerably higher than that of Group II, indicating that high nutrient (ammonia) pollution led to biotic homogeneity. Overall, turnover was the key factor determining the total beta diversity of the two groups, suggesting the key importance of replacement of zooplankton assemblages. Furthermore, we found a correlation between environmental factors (mainly nitrogen content) and the beta diversity of zooplankton, and beta diversity (Brepl and Btotal) decreased with increasing trophic state. These findings provide further insight into the changing characteristics of the beta diversity patterns of zooplankton in river networks and may help to guide managers dealing with conservation strategies for aquatic biodiversity preservation in urban river ecosystems.