Plethodontid salamanders of the genusDesmognathusexhibit varying levels of genetic differentiation among and within both allopatric and sympatric taxa. This provides excellent opportunities to study population differentiation and speciation. Two morphologically similar species in this genus,D. imitatorandD. ochrophaeus,are genetically well‐differentiated from one another and occur in sympatry with no evidence of hybridization and introgression. We report that the degree of sexual isolation between these two species is very high, regardless of whether the populations under comparison are allopatric or sympatric with one another. Neither reinforcement nor reproductive character displacement are required to explain the evolution of sexual incompatibility in sympatry. Sexual behaviour apparently diverges while populations are allopatric with one another. Preliminary study indicates thatD. imitatorconsists of populations among which there may be significant sexual isolation in the absence of detectable genetic differentiatio