Some unisexual invertebrates engage in low levels of sex with related sexual species, and this raises the possibility that unisexual vertebrates have a similar capability. In this study, I compared the population genetic signatures of a triploid population of unisexual salamanders in the genus Ambystoma and sympatric sexual A. jeffersonianum to investigate the degree to which clonal reproduction is occurring in this natural unisexual population. Analysis of nine microsatellite loci revealed extremely high allelic and genetic diversity in both the A. jeffersonianum and triploid populations, indicating that strict clonal reproduction is unlikely to be occurring among these unisexuals. In contrast, significant linkage disequilibrium for all pairs of loci, high heterozygote excess, and large variation in F_IS values across all microsatellite loci (range of F_IS  =  −0.710 to −0.157) in the triploid population indicate that gynogenesis is the most common but not exclusive form of reproduction occurring in unisexual Ambystoma. This study provides the best evidence thus far that unisexual Ambystoma are utilizing a complex mode of reproduction that has features of both sexual and asexual reproduction.