|Being lungless, plethodontid salamanders respire through their skin and are especially sensitive to environmental disturbances. Habitat fragmentation, low abundance, extreme habitat requirements, and a narrow distribution of less than 70 miles in length, makes one such salamander, Plethodon punctatus, a species of concern (S1) in West Virginia. To better understand this sensitive species, day and night survey hikes were conducted through ideal habitat and coordinate data as well as tail tips (10 to 20 mm in length) were collected. DNA was extracted from the tail tips and polymerase chain reaction (PCR) was used to amplify mitochondrial 16S rRNA gene fragments. Maximum parsimony, neighbor-joining, and UPGMA algorithms were used to produce phylogenetic haplotype trees, rooted with P. wehrlei. Based on our DNA sequence data, four disparate management units are designated. Surveys revealed new records on Jack Mountain, a disjunct population that expands the known distribution of the species 10 miles west. In addition, surveys by Flint verified a population on Nathaniel Mountain, WV and revealed new records on Elliot Knob, extending the known range several miles south. DNA sequencing of 24 individuals revealed 8 haplotypes. 16 individuals from the main population on Shenandoah Mountain all had the same haplotype, suggesting low genetic variability. Conversely, each individual from all other areas possessed a unique haplotype. Most importantly, a haplotype from Nathaniel Mountain, WV was deeply divergent and has probably been isolated since the early Pleistocene, making the population a conservation priority. It is hoped that this new genetic data will increase the efficacy of Cow Knob salamander conservation efforts by providing the means to implement management plans that conserve intraspecific genetic diversity.