A hierarchical clustering technique was used to classify sites in the upper Tennessee River basin based on relative abundance of fish species. Five site groups were identified. These groups differed mainly by the occurrence of minnow and darter species. Drainage area and ecoregion were the primary factors explaining the differences among groups; elevation and human influence were of secondary importance. The Blue Ridge site group had the most distinct fish assemblage, large-river sites supported the highest species richness, and the upper Ridge and Valley sites contained the highest percentages of environmentally sensitive species. These results can provide a regional framework for management and conservation activities.