Described in 2009, the Patch-nosed Salamander (Urspelerpes brucei) is a miniature species of lungless salamander with a geographic range of only ~45 km². This species is endemic to the foothills of the Appalachian Mountains in extreme northeastern Georgia and northwestern South Carolina. The Tugaloo River—a waterway of some 50 m in width that forms the political boundary between the two states—bisects the tiny range of U. brucei and likely acts as a barrier to gene flow. Using RADcap data and a suite of complementary population genomic analyses, we evaluated the role that this river and its tributaries may play in enabling and/or interrupting gene flow among populations of U. brucei, and we investigated patterns of within-population and between-population genetic variation. Our results revealed a general pattern of isolation-by-stream distance and indicated that a population separated by the Tugaloo River is moderately more differentiated than what is explainable by stream distance alone. Unique in both its physiography and geologic history, this region in which U. brucei lives also harbors more than a dozen other species of lungless salamanders. Therefore, the genetic patterns that we have elucidated may have larger implications for differentiation among populations of other species with similar dispersal abilities.