AbstractUnderstanding how and why fish species composition varies between locations through beta‐diversity has received increased interest recently. The physical structure, extension and environmental characteristics along the river basins influence the dispersal of organisms, which ultimately affects metacommunity dynamics and biodiversity patterns. Here, we aimed to identify major breaks delimiting fish assemblages in a large dam‐free river basin on its main channel, which is almost pristine in hidrology, and to evaluate the influence of geo‐climatic variables in the observed beta‐diversity patterns. We used the statistical techniques Species Composition Interpolation (SCI) and Generalized Dissimilarity Modeling (GDM) to analyse beta‐diversity patterns based on georeferenced fish records from the Ivaí River basin. Ichthyofauna units were delimited, estimating the association between geo‐climatic predictors and beta‐diversity. We identified three major breaks delimiting fish assemblages in the Ivaí River basin (i.e. the upper, middle and lower sections). The fish communities were very distinct at the upper and lower sections, while the middle section was recovered as less differentiated. Turnover was the main component contributing to total beta‐diversity, and dissimilarity in fish species composition was moderately associated with geo‐climatic variables. Fish communities from the Ivaí River basin must be associated with niche‐based processes, and we hypothesize that dispersal processes can be equally significant at the basin scale. The investigations may produce insights on strategies for conservation and restoration of degraded rivers, contributing to the long‐term maintenance of fish communities and to the delimitation of new strategic areas for the effective conservation of freshwater biodiversity.