Nonwadeable rivers are unique ecosystems that support high levels of aquatic biodiversity, yet they have been greatly altered by human activities. Although riverine fish assemblages have been studied in the past, we still have an incomplete understanding of how fish assemblages respond to both natural and anthropogenic influences in large rivers. The purpose of this study was to evaluate associations between fish assemblage structure and reach-scale habitat, dam, and watershed land use characteristics. In the summers of 2011 and 2012, comprehensive fish and environmental data were collected from 33 reaches in the Iowa and Cedar rivers of eastern-central Iowa. Canonical correspondence analysis (CCA) was used to evaluate environmental relationships with species relative abundance, functional trait abundance (e.g. catch rate of tolerant species), and functional trait composition (e.g. percentage of tolerant species). On the basis of partial CCAs, reach-scale habitat, dam characteristics, and watershed land use features explained 25.0–81.1%, 6.2–25.1%, and 5.8–47.2% of fish assemblage variation, respectively. Although reach-scale, dam, and land use factors contributed to overall assemblage structure, the majority of fish assemblage variation was constrained by reach-scale habitat factors. Specifically, mean annual discharge was consistently selected in nine of the 11 CCA models and accounted for the majority of explained fish assemblage variance by reach-scale habitat. This study provides important insight on the influence of anthropogenic disturbances across multiple spatial scales on fish assemblages in large river systems.