The fishes of the present study were collected in the headwater streams of the Sorocaba, Paranapanema and Ribeira de Iguape river basins during the dry period in 2010. A total of 2892 fishes, grouped in 53 species, were captured. The composition of the ichthyofauna captured in the streams of Sorocaba and Paranapanema river basin was greatly similar. On the other hand, the fish fauna of the streams of Ribeira de Iguape river basin were quite different from the ones captured in the others basins, with the occurrence of endangered species (Isbrueckerichthys epakmos and I. duseni) and exotic species (Misgurnus anguillicaudatus). The previous list of fish for the Sorocaba river basin increased with the addition of seven species of Characiformes, one Gymnotiformes and four Siluriformes.

Understanding spatial patterns of species diversity and the variables that structure biological communities is critical to successful ecosystem management. Regional diversity (γ) can be considered a combination of local diversity (α) and among sites variation (β). Using abundance data of fish species in 19 stream stretches, an analysis of diversity partitioning was used to determine the contribution of α‐ and β‐diversity to γ‐diversity. Redundancy analysis was applied to find the contribution of environmental variables and spatial configuration to species composition. Intersite variation contributed significantly to γ‐diversity. Spatial configuration and instream heterogeneity (coarse substrate, channel width, water velocity and shading) and riparian vegetation were related to local fish abundances. Conservation actions should consider that all streams are important, and prioritisation of just a small number of the richest sites is not appropriate.

Disentangling the hierarchical structure of river systems and multi-scale environmental effects is essential for understanding freshwater fish community structure and function. We sampled 70 stream stretches to investigate how the taxonomic and functional composition and diversity of fish assemblages responded to the environment considering a hierarchical organisation of streams in three Brazilian watersheds (Upper Sorocaba, Upper Paranapanema and Upper Ribeira de Iguape). Functional diversity indicated that stream fish community function was independent of the watershed. The most critical environmental variable was substrate gradient. In streams with a consolidated substrate, equitability and functional dispersion were low due to invertivores occurring in this type of substrate. Our results highlight the importance of local environmental filters for shaping streams fish assemblages across different basins. This shaping was more evident when functional descriptors of assemblages were analysed, thereby reinforcing the importance of an approach based on traits as a tool to elucidate local community assembly processes at broader spatial scales.