Resumo. Abstract: Aquatic ecosystems have a great diversity of habitats, including streams that are of extreme ecological and economic importance but have undergone impacts, such as the input of domestic and industrial waste and deforestation of riparian forest. However, few studies have been carried out on the diversity, specifically beta diversity, of fish assemblages in urban and rural streams. Aim In this context, the objective of this work was to verify if there are differences in fish assemblage structure between urban and rural streams, with focus on beta diversity. Methods We selected 12 streams, 6 urban and 6 rural, located in the municipalities of Ourinhos (SP) and Jacarezinho (PR) in the Paranapanema river basin. Fishes were collected using a sieve. Concomitantly water quality variables were also measured, such as temperature, pH, conductivity and total dissolved solids. To assess whether fish beta diversity differed between streams, a dispersion homogeneity test (Permutational Analysis of Multivariate Dispersions) was calculated. Results Significant differences were observed in conductivity and total dissolved solids, both with higher values for urban streams. In urban streams, 12 species of fish were identified, five of which were exclusive, while in rural streams, 18 species were recorded, ten of which were exclusive. Furthermore, both composition and beta diversity were significantly different between rural and urban streams, with the highest values recorded in rural streams. Conclusions This study provides evidence of how urbanization impacts the composition and beta diversity of fish in streams and highlights the importance of sustainable management such as effluent control, restoration of riparian vegetation, and the determination of protection areas.