Nearly 168 species of Mammillaria are endangered at the global level; most of these cactus species exhibit a narrow distribution range and/or small population size. Recently, the first population genetics studies showed low to moderate levels of heterozygosity in this genus. This study examines Mammillaria huitzilopochtli and M. supertexta, 2 threatened species endemic to the central semiarid region of Mexico, to propose conservation actions, as well as to identify the underlying processes that determine the levels of population genetic diversity in these species. A total of 106 and 148 individuals from 5 populations were sampled for M. huitzilopochtli and M. supertexta, respectively. Each individual was genotyped at 8 microsatellite loci. The levels of heterozygosity were high in the 2 species, but differences in allelic richness were detected. Genetic differentiation between populations was significant in both species, with evidence for isolation by distance in M. supertexta but not in M. huitzilopochtli. We postulated that genetic drift; geographic isolation and inbreeding are the most important processes shaping the populations' genetic variability and differentiation. We identified 3 and 4 genetic groups for M. huitzilopochtli and M. supertexta, respectively. In the Tehuacán-Cuicatlán valley these groups may be used as references to guide plant conservation efforts under ecosystem-based approach.