Understanding biodiversity patterns and their drivers across large spatial scales has become a necessary component of addressing complex multiscale conservation challenges. Increasingly, functional diversity measures are being used to provide insight on the ecological integrity of ecosystems. We estimated functional diversity at two different spatial extents using data sets of contrasting sampling intensity and time duration and compared these two different approaches. For our regional‐scale analysis, we used large‐scale fish sampling data sets and identified 397 species across subbasins within the southeastern USA. For our state‐scale analysis, we used data from the South Carolina Department of Natural Resources Statewide Stream Assessment through which 101 species were collected from wadeable streams across South Carolina. For all fish species, we scored trophic and reproductive traits and measured trophic and reproductive functional diversity across subbasins and South Carolina streams. We assessed differences in functional diversity between level III ecoregions, quantified the influence of imperiled and invasive species on functional diversity, and modeled relationships between functional diversity and environmental variables at the state scale using Random Forest regression. At both scales, we found support for higher trophic functional diversity in high‐elevation ecoregions, while reproductive functional diversity was generally higher in low‐elevation ecoregions. The inclusion of imperiled species increased functional diversity estimates, while invasive species decreased functional diversity estimates except for reproductive functional diversity at the regional scale. Environmental variables that correlated with functional diversity at the state scale included forest cover, elevation, and conductivity. The observed spatial patterns of functional diversity, the environmental variables identified as predictors of functional diversity, and knowledge of how imperiled and invasive species influence functional diversity can be used to guide freshwater fish conservation efforts across the southeastern U.S. region. Additionally, these findings highlight the utility of data from intensive, standardized, broad‐scale aquatic surveys in addressing these and similar questions regarding freshwater diversity.