In reservoir cascades, effects on fish assemblages are expected to strengthen over time and transfer from one reservoir to an adjacent one. To test this, fish assemblage data from 23 years of monitoring in the Araguari reservoir cascade system, upper Paraná River basin, were analyzed. From 1993 to 2015, 72 fish species were recorded in the studied reservoirs, representing five orders and 19 families. In all, 58 species were native to the basin and 14 non-native (10 from other Brazilian basins and four from other countries). The results showed a clear reduction in richness of native and migratory fish species and an increase of non-native species, following reservoir formation. Migratory species richness was higher in reservoirs that presented habitats similar to lotic stretches or tributaries upstream of the impounded area. A clear tendency for native species’ decline and non-native fish abundance to increase, was observed in a downstream direction. Fish assemblages became increasingly dissimilar as reservoirs became more distant from each other (longitudinal gradient) and were dominated by small and medium sized species. Alongside biogeographic factors and the presence of non-piscivorous non-native species, reservoir area and age were found to be important drivers of variation in fish assemblage structure. Results from this study help to clarify the potential accumulated impacts of reservoirs cascades on fish diversity, which must be carefully considered in river basin inventories for hydropower plants, and reinforces the importance of long-term monitoring, considering longitudinal and lateral dimensions of the basin.