Extensive genetic variation has been documented throughout the range of the Two-lined salamander (Eurycea bislineata) species complex; however, a lack of fine-scale studies across putative species boundaries in this group has made it difficult to ascertain the amount of gene exchange that occurs between different genetic groups. We investigated patterns of gene exchange in populations from the Blue Ridge Mountains and adjacent Atlantic Slope, by analyzing allozyme variation along a transect that traverses a hypothesized contact zone between montane and lowland forms of the complex, E. wilderae and E. cirrigera. Geographically concordant transitions in allele frequencies at four allozyme loci indicate that a contact zone between these forms exists in the Savannah River drainage in the Piedmont of South Carolina. Variation in the PEP-B locus exhibits a broad cline that crosses the transition between the Blue Ridge and Piedmont physiographic provinces. A fixed difference at one locus (PK), nearly fixed differences at two other loci (AAT-1 and PEP-LA), and a Nei's genetic distance of 0.22 (D = 0.17–0.32) across the contact zone reflect an extended history of reduced gene exchange between these forms. The genetic data from the contact zone suggest that these montane and lowland forms are distinct species.