The loss of genetic diversity in small or isolated populations can increase inbreeding, decrease fitness and adaptive potential and increase a species’ probability of extinction. In species with life histories that naturally result in small populations and/or low levels of gene flow, patterns of anthropogenically induced genetic erosion can be obscured by evolutionary history; yet these species may still be susceptible to genetic loss. We assess genetic diversity among populations of Ambystoma salamanders endemic to Mexico, including populations that are facultatively or obligately paedomorphic, to test whether paedomorphic lineages have lower genetic diversity than metamorphic ones, and whether gene flow contributes to the maintenance of diversity in divergent forms with either life history. We also test the utility of our markers in assigning illegally harvested individuals to populations of origin. We found reduced genetic diversity in paedomorphic compared with some, but not all, metamorphic populations. Populations of both forms showed genetic signatures of bottlenecks, underscoring that factors other than paedomorphosis contribute to historical reductions in population size. In general, Ambystoma populations have low interpopulation gene flow and admixture, but paedomorphic populations have higher within-population relatedness than most metamorphic populations. We discuss historical and current landscape attributes that impact populations and their connectivity, the implications of our findings for ongoing captive propagation programs and the prospects for continued genetic health of Ambystoma in México.