The Eurycea bislineata (Two-lined salamander) species complex is characterized by extensive geographic genetic fragmentation throughout its range. I tested for sexual isolation and divergence in courtship behavior across a contact zone between E. wilderae and E. cirrigera. I found concordance between patterns of geographic genetic fragmentation and sexual isolation that suggests that divergence in the mate-recognition systems of E. cirrigera and E. wilderae prevents free gene exchange across their contact zone. In addition, the courtship behavior of the E. bislineata complex provides insights into alternative hypotheses of evolution of the mental gland and pheromone delivery behavior in plethodontid salamanders.