Abstract Aim The alteration of flowing systems over the past century has led to significant changes to the processes that drive these complex environments as well as to the scales at which these processes act. Recently, efforts have begun in earnest to restore some semblance of ecosystem diversity, but there is little understanding of exactly on what spatial scale or scales biotic diversity is responding. We investigated the manner in which fish diversity is partitioned at multiple spatial scales in two rivers in the central United States. Location The Missouri and Illinois rivers of the central United States. Methods We analysed how fish diversity was partitioned within the Illinois River and Missouri River systems by sampling each river under hierarchical frameworks that allowed analysis at section (large), reach (intermediate), and site (small) scales. We tested the hypothesis that there are scale-dependent responses of fish diversity using an additive partitioning approach. Results Site alpha diversity was significantly higher than expected in both the Illinois and Missouri rivers. The relative contribution of alpha diversity to total diversity at a given spatial scale increased for the Illinois River, but not for the Missouri River, in that the highest alpha diversity contribution peaked at the reach scale. Diversity patterns from both rivers suggest that diversity at the site scale plays a significant role in determining the overall diversity in these systems. However, there is a substantial contribution at larger scales that warrants consideration when attempts are being made to protect or restore diversity and other ecosystem parameters. Main conclusions Understanding the variation of diversity in riverine systems is crucial for providing insight not only into how biotic communities respond to scale-dependent factors, but also into the underlying abiotic and biotic factors that generate patterns of diversity across scales. These insights, in turn, are important for ensuring that restoration and management activities are targeting the appropriate scales for remediation. A lack of understanding of this issue could have negative outcomes for the recovery of a community in a restoration scenario, as well as resulting in a low economic return on restoration investments, which could hinder future efforts.