I tested if spatial variation of Ohio River drainage basin fish assemblages, current habitat, water quality, and hydrology were concordant with historic glaciation boundaries. Assemblage analyses were organized using taxonomic and functional groups. Multivariate analyses, principal components analysis (PCA) and indicator species analyses, were used to test whether taxonomic and functional assemblages were distinct among regions with varying glacial histories. Principal components analysis was used to identify habitat and water quality as well as hydrologic gradients that could be discerned by glacial region. I identified significant differences in both taxonomic and functional fish assemblage structure and habitat variation among regions that had different glaciation histories. Recently glaciated Wisconsinan sites were characterized by assemblages of Cyprinidae and Catostomidae families, with high abundances of tolerant fishes that tended to occur in habitats with reduced current velocity. Sites in the Pre- Wisconsinan region were characterized by Cyprinidae, Catostomidae, Centrarchidae, and Percidae families, with increased abundances of intolerant fishes that tended to occur in habitats with coarser substrates and increased water velocity in streams of varying size. Sites in the unglaciated region were dominated by Cyprinidae and Percidae families but were not closely associated with any habitat-based functional group. Taxonomic and functional analyses explained 25% and 40%, respectively, of the variance in assemblage structure. Mean habitat PCA component scores in the unglaciated and Pre-Wisconsinan sites were significantly different than Wisconsinan sites, which were characterized by increased channel structure and reduced stream size. While a multitude of factors impact the distribution of fishes, these results suggest that historical influences such as glaciation may be used to further explain underlying mechanisms of spatial variation in fish assemblages. / Department of Biology