We studied the leading causes of the spatial distribution pattern of fish species assemblage in the transition region between Cerrado-Caatinga biomes, in the São Francisco River (Brazil). Biotic and abiotic variables were collected at 17 sampling sites located in three sub-basins, in two periods during 2015. Some 1689 individual fish were sampled, distributed in 69 species, 51 genera, 22 families and seven orders. There was a low overlap of species, with only seven species in common among the three sub-basins. The most substantial degree of sharing occurred between sub-basins from the Cerrado biome, with 22 restricted species. Crenicichla lepidota, Psellogrammus kennedyi, and Hoplosternum littorale were associated with the sub-basin from Caatinga, and Astyanax aff. eigenmanniorum and Bryconops aff. affinis with Cerrado sub-basins. The fish assemblage showed significant spatial variation between biomes, and the distribution was determined by an interaction of regional altitude variable with local variables such as river width, substrate and water velocity (environmental factors), but geographical factors were also important. Fish assemblage difference along a large river course has significant implications for conservation strategies, management or evaluation of biodiversity, needing several strategies for their preservation considering small geographic areas.