A common minnow in headwaters of many eastern United States river systems, the rosyside dace (Clinostomus funduloides) is often considered an indicator species related to health of river ecosystems. While abundant in most river basins in western North Carolina, such as the Catawba and Broad basins, the rosyside dace has only recently been discovered within the Upper French Broad (UFB) basin. Though preliminary data have suggested that at least one population may be endemic, questions remain regarding the origin of this and other populations. To address these, DNA was isolated from muscle or liver tissue of specimens collected from sites in the UFB, Catawba, and Broad river basins. The cyt-b gene, a highly conserved region of mitochondrial DNA, was then PCR-amplified and sequenced. Using GenBank BLAST, these sequences were compared to identify potential relationships between populations. While data support the hypothesis that rosyside dace were recently introduced to the UFB from recorded populations in the Catawba basin, some UFB specimens share mutations suggesting introduction from other sources, or evolutionary divergence from an ancestral population predating the formation of the Eastern Continental Divide. Given the current paucity of genetic data both from recorded and newly discovered populations within the southern Appalachian region, this research provides original data suggesting likely origin of the UFB populations, and offers possible evolutionary relationships of all regional populations examined. Confirming the existence of endemic rosyside dace populations within the UFB will inform further study of their range, any necessary conservation, and policy regarding maintenance of riparian and river ecoystems within the region.