We used three diagnostic protein markers to examine salamanders of the Eurycea bislineata complex at 80 localities in Virginia and West Virginia. Two groups were strongly differentiated and met at a narrow contact zone. Rare hybridization was observed as well as limited introgression up to 5 km north and 10 km south of the contact zone. At the contact zone, 1% F1, 2% F2, 32% backcross, and 66% parental genotypes were observed. This pattern of parapatric distribution with limited hybridization and introgression argues for the recognition of Eurycea bislineata and E. cirrigera as separate species.