The woodland and dusky salamanders (Plethodon and Desmognathus) of the southern Appalachians show how molecular studies can deepen our understanding of regional biodiversity. They also show how technological developments can drive shifts in focus, in this case from allele frequencies and population genetics to nucleic acid sequences and phylogenetic inference. This shift has left unanswered important questions about the processes that underlie diversification. Relationships between genetic differentiation and geographic separation provide insight into such questions. Isolation by distance (IBD) relationships in Plethodon and Desmognathus indicate that genetic distance increases more rapidly with geographic separation from north to south in the Appalachian Mountain region of eastern North America. In Desmognathus, genetic divergence appears to increase faster with geographic separation than in Plethodon, perhaps as a consequence of isolation between oviposition sites and larval habitats. Reproductive compatibility may also be retained at higher levels of genetic divergence in Desmognathus than in Plethodon. These patterns deserve further exploration in these and other species, as sequencing techniques are applied in studies of allelic variants at nuclear loci.