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Abstract We assessed the fish assemblage structure and composition of Nova Ponte Reservoir (Araguari River, Upper Paraná Basin, Brazil). We observed significant differences in abundance (p = 0.0003), richness (p = 0.0005) and diversity (p = 0.02) between lacustrine and riverine zones of the reservoir. Nine species were significantly more abundant in the riverine region: Astyanax altiparanae, Astyanax gr. fasciatus, Galeocharax knerii, Hoplias intermedius, Hypostomus sp., Leporinus friderici, Leporinus obtusidens, Pimelodus maculatus and Schizodon nasutus. The results indicated a longitudinal gradient in the composition and abundance of fishes in Nova Ponte Reservoir, reinforcing the importance of freely flowing riverine reaches for conserving native neotropical ichthyofauna and reflecting the strong adaptation of these species to riverine systems.