Abstract The structure of freshwater assemblages may be driven directly by urbanization or indirectly by a reduction in environmental heterogeneity (EH). Disentangling the effects of urbanization and EH requires uncorrelated proxies of each of these factors. We assessed the effects of the degree of urbanization and EH on the structure of fish assemblages. We sampled fish in 45 streams located in the urban area of Cuiabá. We assessed the effects of urbanization and EH on rarefied fish species richness (Srarefied), the local contribution to beta diversity (LCBD), and composition with linear models and distance-based redundancy analysis. Our indexes of urbanization and EH were not correlated. We found that both Srarefied and the LCBD decreased with an increasing degree of urbanization, but were not associated with EH. We also noted that few native fish species abundances were associated with the EH. Serrapinnus microdon, S. calliurus, Hemigrammus tridens, and Astyanax lacustris were abundant in streams with a lower degree of urbanization. The non-native Poecilia reticulata was more abundant in streams with a higher degree of urbanization. Our results highlight that urbanization leads in negative impacts on fish assemblages, such as decreases in diversity and the dominance of non-native species.