Abstract. Abstract Aim: Heterogenous environments can contribute to maintain biodiversity. Traditionally beta diversity studies have focused on determining the effect of environmental variables on the total dissimilarity of species composition. However, decomposing beta diversity in species replacement and nestedness could give new insights on mechanisms affecting spatial patterns of biodiversity. We aimed to answer two main questions about spatial patterns of fish diversity in a Neotropical basin: 1) whether some regions contribute differently to fish diversity, and 2) whether species turnover and nestedness are explained by environmental gradients. Methods Sampling sites in the main channel and tributaries of the Upper Paraná River were sampled between 2013 and 2015. We partitioned beta diversity and tested the relationship of turnover and nestedness with environmental variables. Results 74 species were captured. Some of these species were restricted to different sites, contributing to variation in species composition. Hill numbers showed a trend for higher diversity in the tributaries than in Paraná River sampling sites, and the partition of beta diversity revealed that species replacement drove dissimilarity in species composition. Only total beta diversity and turnover were related to environmental variables, mainly conductivity and turbidity. Conclusions Species diversity and composition of fish assemblages in the Upper Paraná River could be related to environmental gradients. Overall, our results suggest that Paraná River tributaries contribute to increase environmental heterogeneity, and hence to maintain a high diversity and variation in species composition. For that reason, we strongly recommend preserving highly heterogeneous habitats in the region.