1. We examined the effects of physical and chemical habitat variables and ecoregions on species occurrence and fish assemblage structure in streams of the Paraíba do Sul basin, in southeast Brazil. 2. Fish and environmental data were collected from 42 sites on 26 first to fourth order streams (1 : 50 000 map scale) in three ecoregions. The sites occurred in one valley and two plateau ecoregions at altitudes of 40-1080 m and distances of 0.1-188 km from the main channel of the Rio Paraíba do Sul. Physical habitat (substratum, riparian cover, habitat types) and water quality (dissolved oxygen, pH, temperature and conductivity) variables were measured at each sampling site. 3. A total of 2684 individuals in 16 families and 59 species were recorded. 4. Ecoregion was a better predictor of the fish assemblage than the other environmental variables, according to the differences between the mean within-class and mean betweenclass similarities in assemblage data. 5. Differing landscape characteristics were associated with differing local variables and thereby with differing fish assemblage structures. Riffles, shrub, grass, dissolved oxygen, conductivity and temperature were closely related to fish assemblage structure. 6. Fish assemblages in sites far from the main river and at higher altitudes also differed from those near the Paraíba do Sul main channel, presumably as a result of differences in connectivity, covarying environmental factors and anthropogenic influence. 7. These results reinforce the importance of understanding how stream communities are influenced by processes and patterns operating at local and regional scales, which will aid water resource managers to target those factors in their management and rehabilitation efforts.