Understanding trends in the diverse resources provided by large rivers will help balance tradeoffs among stakeholders and inform strategies to mitigate the effects of landscape scale stressors such as climate change and invasive species. Absent a cohesive coordinated effort to assess trends in important large river resources, a logical starting point is to assess our ability to draw inferences from existing efforts. In this paper, we use a common analytical framework to analyze data from five disparate fish monitoring programs to better understand the nature of spatial and temporal trends in large river fish assemblages. We evaluated data from programs that monitor fishes in the Colorado, Columbia, Illinois, Mississippi, and Tallapoosa rivers using non-metric dimensional scaling ordinations and associated tests to evaluate trends in fish assemblage structure and native fish biodiversity. Our results indicate that fish assemblages exhibited significant spatial and temporal trends in all five of the rivers. We also document native species diversity trends that were variable within and between rivers and generally more evident in rivers with higher species richness and programs of longer duration. We discuss shared and basin-specific landscape level stressors. Having a basic understanding of the nature and extent of trends in fish assemblages is a necessary first step towards understanding factors affecting biodiversity and fisheries in large rivers.