Amphibians are globally threatened by habitat loss and fragmentation; species within the order Ambystoma are not the exception, as there are 18 species of mole salamanders in México, of which 16 are endemic and all species are under some national or international status of protection. The mole salamander, Ambystoma altamirani is a microendemic species, which is distributed in central México, within the trans-Mexican volcanic belt, and is one of the most threatened species due to habitat destruction and the introduction of exotic species. Nine microsatellite markers were used to determine the genetic structure, genetic variability, effective population size, presence of bottlenecks and inbreeding coefficient of one population of A. altamirani to generate information which might help to protect and conserve this threatened species. We found two genetic subpopulations with significant level of genetic structure (F = 0.005) and high levels of genetic variability (H = 0.883; H = 0.621); we also found a small population size (N = 8.8), the presence of historical (M = 0.486) and recent bottlenecks under IAM and TPM models, with a low, but significant coefficient of inbreeding (F = -0.451). This information will help us to raise conservation strategies of this microendemic mole salamander species.