During the colder climates of the Pleistocene, the ranges of high-elevation species in unglaciated areas may have expanded, leading to increased gene flow among previously isolated populations. The phylogeography of the pygmy salamander, Desmognathus wrighti, an endemic species restricted to the highest mountain peaks of the southern Appalachians, was examined to test the hypothesis that the range of D. wrighti expanded along with other codistributed taxa during the Pleistocene. Analyses of genetic variation at 14 allozymic loci and of the 12S rRNA gene in the mtDNA genome was conducted on individuals sampled from 14 population isolates throughout the range of D. wrighti. In contrast to the genetic patterns of many other high-elevation animals and plants, genetic distances derived from both molecular markers showed significant isolation by distance and genetic structuring of populations, suggesting long-term isolation of populations. Phylogeographical analyses revealed four genetically distinct population clusters that probably remained fragmented during the Pleistocene, although there was also evidence supporting recent gene flow among some population groups. Support for isolation by distance is rare among high-elevation species in unglaciated areas of North and Middle America, although not uncommon among Plethodontid Salamanders, and this pattern suggests that populations of D. wrighti did not expand entirely into suitable habitat during the Pleistocene. We propose that intrinsic barriers to dispersal, such as species interactions with other southern Appalachian plethodontid salamanders, persisted during the Pleistocene to maintain the fragmented distribution of D. wrighti and allow for significant genetic divergence of populations by restricting gene flow.