Abstract Dams reduce the longitudinal connectivity of rivers and thereby disrupt fish migration and the spatial distribution of species, impacts that remain poorly studied for some Neotropical rivers from mega-diverse basins. We investigated the spatial distribution of fish species with different trophic and movement/reproductive/size characteristics to assess how functional groups have responded to a cascade of dams on the Uruguai River in southern Brazil. Fish abundance, biomass, and species composition were evaluated at eight locations along the longitudinal gradient. The fish assemblage in the upper stretch was mainly characterized by small and medium-sized species at higher trophic levels, whereas the sites located furthest downstream displayed more medium and large-sized species, including many carnivorous species. Species with high fecundity, seasonal migrants, and catfishes with internal fertilization were common in the river´s middle and lower reaches. Detritivorous species dominated areas distant from the dams. Overall, functional diversity of local fish assemblages was greater in lower reaches. The cascade of dams has impacted the distribution of functional groups of local fish assemblages of Uruguai River. The alteration of functional groups in upper reaches of the river has potential consequences for ecosystem processes and services, such as nutrient cycling and fisheries.