Fish species’ occurrence is influenced by environmental conditions across multiple spatial and temporal scales. Alteration to stream systems has the potential to affect occurrence patterns of fish species. Stream systems of the North America Great Plains face threats originating from climate change and anthropogenic activities resulting in negative implications for many fish species. The Sandhills Ecoregion, Nebraska is considered one of the largest intact grasslands in North America, and thus may experience fewer threats than surrounding areas. Stream systems of the Sandhills Ecoregion are home to 12 fish species of greatest conservation need (SGCN). Little context exists regarding the environmental associations of fish SGCN and, to our knowledge, no studies have focused evaluations on how fish assemblages have changed through time in the Sandhills Ecoregion. Therefore, this study sought to 1) identify the multiscale environmental associations of fish SGCN inhabiting Sandhills Ecoregion streams using occupancy models and 2) assess how fish assemblages have changed since statewide sampling in 1939-1941 using retrospective analyses. We sampled 95 sites for fish assemblages and environmental data throughout the Niobrara River and Elkhorn River drainages of the Sandhills Ecoregion. We also built a database of historical fish occurrence since 1939 for the Niobrara River and Elkhorn River drainages of the Sandhills Ecoregion. Occupancy models constructed using contemporary survey data highlighted that fish SGCN occurrence is low. We also identified that fish SGCN occurrence is mediated by hierarchical multiscale environmental factors. Common variables included in top-ranked models were depth-to-groundwater, index of watershed integrity, stream slope, and proportion of predators. We also identified that assemblages of the Sandhills Ecoregion have exhibited shifts in the relative frequencies of some fish species, but have experienced limited shifts in relative frequencies at the functional trait level. Results from this study provide managers with a rigorous baseline of fish species occurrence in the Sandhills Ecoregion from which to compare to future assessments and may be useful when determining conservation actions.