The plethodontid salamander Desmognathus orestes, a member of the D. ochrophaeus species complex, is distributed in southwestern Virginia, eastern Tennessee, and western North Carolina. Previous allozyme analyses indicate that D. orestes consists of two distinct groups of populations (D. orestes 'B' and D. orestes 'C') with extensive intergradation and probable gene flow between these two groups. Spatially varying allele frequencies can reflect historical associations, current gene flow, or a combination of population-level processes. To differentiate among these processes, we use multiple markers to further characterize divergence among populations of D. orestes and assess the degree of intergradation between D. orestes 'B' and D. orestes 'C', specifically investigating variation in allozymes, mitochondrial DNA (mtDNA), and reproductive behavior among populations. On a broad scale, the mtDNA genealogies reconstruct haplotype clades that correspond to the species identified from previous allozyme analyses. However, at a finer geographic scale, the distributions of the allozyme and mtDNA markers for D. orestes 'B' and D. orestes 'C' are discordant. MtDNA haplotypes corresponding to D. orestes 'B' are more broadly distributed across western North Carolina than predicted by allozyme data, and the region of intergradation with D. orestes 'C' indicates asymmetric gene flow of these markers. Asymmetric mating may contribute to observed discordance in nuclear versus cytoplasmic markers. Results support describing D. orestes as a single species and emphasize the importance of using multiple markers to examine fine-scale patterns and elucidate evolutionary processes affecting gene flow when making species-level taxonomic decisions.