The behavioral basis of sexual isolation was investigated in 11 crosses staged between individuals from nine allopatric populations of the Appalachian mountain dusky salamander, Desmognathus ochrophaeus. Scan-sampled observations of within-population (homotypic) and between-population (heterotypic) heterosexual encounters yielded the following results. 1) Fewer heterotypic encounters than homotypic encounters resulted in insemination of females. Indices of the strength of sexual isolation based on insemination data ranged from 0.26 (lowest) to 0.82 (highest), indicating that mechanisms that confer sexual isolation may evolve during allopatric differentiation. 2) Heterotypic encounters that progressed as far as the stage of courtship during which the male stimulates the female to mate did so at the same time as homotypic encounters, although the number of the former reaching this stage was lower. 3) The time interval from courtship initiation to insemination was longer in heterotypic encounters than homotypic encounters. In some crosses, males did not attempt to initiate heterotypic encounters, indicating that this sex is responsible for sexual isolation in these instances. We use our results to formulate specific hypotheses concerning the behavioral basis of sexual isolation in these salamanders, and some possible experimental approaches are suggested.