Anthropogenic impacts on the landscape can drive biotic homogenization whereby distinct biological communities become more similar to one another over time. Land-use change in the Southern Appalachian region of the United States is expected to result in homogenization of the highly diverse freshwater fish communities as in-stream habitat alterations favor widespread cosmopolitan species at the expense of more narrowly distributed highland endemic species. We compiled four datasets spanning 25 years to (1) evaluate the effects of environmental factors on relative abundance and richness of highland endemic vs. cosmopolitan species in this region and (2) test for taxonomic homogenization, measured as a change in beta diversity over time. We found that several environmental factors differentially affected highland endemic and cosmopolitan species, with the proportion of forested land cover in a watershed most strongly predicting higher relative abundance and richness of highland endemic species. Our analysis of beta diversity change, however, shows mixed evidence of taxonomic homogenization, depending on how common species are weighted. Shifts in community composition, with or without homogenization, may warrant attention in biodiversity conservation planning.