Spatial patterns in assemblage structures are generated by ecological processes that occur on multiple scales. Identifying these processes is important for the prediction of impact, for restoration and for conservation of biodiversity. This study used a hierarchical sampling design to quantify variations in assemblage structures of Brazilian estuarine fish across 2 spatial scales and to reveal the ecological processes underlying the patterns observed. Eight areas separated by 0.7 to 25 km (local scale) were sampled in 5 estuaries separated by 970 to 6000 km (regional scale) along the coast, encompassing both tropical and subtropical regions. The assemblage structure varied significantly in terms of relative biomass and presence/absence of species on both scales, but the regional variation was greater than the local variation for either dataset. However, the 5 estuaries sampled segregated into 2 major groups largely congruent with the Brazilian and Argentinian biogeographic provinces. Three environmental variables (mean temperature of the coldest month, mangrove area and mean annual precipitation) and distance between estuaries explained 44.8 and 16.3%, respectively, of the regional-scale variability in the species relative biomass. At the local scale, the importance of environmental predictors for the spatial structure of the assemblages differed between estuarine systems. Overall, these results support the idea that on a regional scale, the composition of fish assemblages is simultaneously determined by environmental filters and species dispersal capacity, while on a local scale, the effect of environmental factors should vary depending on estuary-specific physical and hydrological characteristics © 2013 Inter-Research.