Green salamanders (Plethodontidae: Aneides aeneus) are rock outcrop habitat specialists, possessing numerous unique morphological adaptations (e.g., prehensile tail and squared toe-pads) for climbing. Some authors believe A. aeneus, which is widely distributed across the Appalachian Mountains of the inland eastern United States, comprises a species complex due to substantial karyotypic variation among populations. We conducted a population genetic and phylogenetic study across the range of A. aeneus and discovered substantial genetic structure, including four distinct lineages, one of which we describe as Aneides caryaensis, new species. Restricted to a narrow geographic distribution in western North Carolina, this species faces pressing conservation threats due to rapid real estate and tourism development in the area. We also recommend the recognition of three geographically distinct and reciprocally monophyletic lineages as evolutionarily significant units due to strong mitochondrial and nuclear differentiation among them. Aneides aeneus has been petitioned for listing under the Endangered Species Act, and our study further highlights the need for conservation management of this complex. Our formal recognition of the extent of genetic and evolutionary diversification of the complex is a critical step in establishing conservation strategies.