Riverine benthic macroinvertebrate (BMI) communities respond to environmental gradients (e.g., climate, land use, and water quality) as well as flow disturbances (e.g., droughts or floods) in a spatially scale-dependent manner. However, research exploring the implications of broader-scale (i.e., regional scale) watershed and environmental gradients on the functional trait composition of BMI communities within and across major river drainages is limited. Understanding the underlying patterns and responses of BMI functional trait composition, site-specific diversity (alpha-diversity), and spatial turnover of traits (beta-diversity) at multiple spatial scales is crucial, particularly considering the anthropogenic effects on landscapes. This study assessed patterns in BMI trait composition and diversity at multiple spatial scales within a single large complex river drainage (Colorado River) and then compared these patterns to those present across several major basins in Texas (Neches, Guadalupe, Red, and Rio Grande). Specifically, we examined the drivers of functional trait composition and diversity at two scales (alpha and beta) as well as the components of beta-diversity (replacement and nestedness) at both spatial scales (within a single drainage versus across basins). Results indicated that the community’s functional trait composition was more dissimilar within all basins except for the Colorado River basin, which was sampled at greater spatial resolution. Functional trait beta-diversity in the Colorado River and Guadalupe basins were driven more by replacement, whereas the Neches, Red, and Rio Grande basins were driven more by nestedness. Overall, our findings indicate that BMI functional trait diversity exhibits substantial spatial scale dependency.