Growing evidence indicates that males may be more discriminating of mating partners than often has been assumed. In the North American Ocoee dusky salamander, Desmognathus ocoee (Plethodontidae: Desmognathinae), sexual incompatibility among conspecific populations is high in encounters staged in the laboratory, at least in part because males fail to recognize ‘other’ females as appropriate targets for courtship. I used Y-mazes to test the hypothesis that males of D. ocoee discriminate between substrate-borne chemical cues produced by ‘own’ (homotypic) and ‘other’ (heterotypic) females. Males of four populations discriminated in favor of substrates soiled by homotypic females over clean (control) substrates (expt 1), suggesting that females produce chemical cues of sociosexual significance to males. Furthermore, males from these populations discriminated in favor of substrates soiled by homotypic females vs. substrates soiled by heterotypic females (expt 2), both conspecific and heterospecific (D. carolinensis and D. orestes). Thus, differences among populations and species in female chemical cues appear to affect the chemotactic responses of males. I suggest that, together with differences in behavioral signals and responses exhibited during courtship, differences in female chemical cues likely contribute to sexual incompatibility among populations and taxa of desmognathine salamanders.