Echinomastus erectocentrus (J.M. Coulter) Britton & Rose var. acunensis (W.T. Marshall) Bravo, the Acuña cactus, is a small, single-stemmed spherical cactus with a restricted distribution across the Sonoran Desert in southern Arizona and into northern Sonora, Mexico. Populations of E. erectocentrus var. acunensis are threatened by loss of habitat, climate change, predation, and border related impacts. Due to the severity of these threats and shrinking population sizes, E. erectocentrus var. acunensis was federally listed as endangered by the United States Fish and Wildlife Service in 2013. The varieties of Echinomastus erectocentrus, E. erectocentrus var. acunensis and E. erectocentrus var. erectocentrus (J.M. Coulter) Britton & Rose, share many morphological characteristics that make them difficult to distinguish from one another. Echinomastus johnsonii (Parry ex Engelm.) E.M. Baxter, a presumed closely related species, also has a high level of morphological overlap that further complicates our understanding of species boundaries and detailed morphological data for these three taxa indicate a geographical cline. The goal of this project is to document the genetic diversity within and among populations of E. erectocentrus var. acunensis, and its close relatives E. erectocentrus var. erectocentrus and E. johnsonii. To accomplish this, populations of E. erectocentrus var. acunensis, E. erectocentrus var. erectocentrus, E. johnsonii and the outgroup Echinomastus intertextus (Engelm.) Britton & Rose were sampled. Deoxyribonucleic acid (DNA) was extracted, and data were collected for nine microsatellite regions developed specifically for these taxa, and two microsatellite regions developed for Sclerocactus, a closely related genus. Standard population genetic measures were used to determine genetic variation and structure, and this observed genetic differentiation was then compared to the current morphological understanding of the group. These analyses help improve the knowledge of the genetic structure of E. erectocentrus var. acunensis and inform the understanding of species boundaries and evolutionary relationships within the group by revealing genetic distinctiveness between all four taxa and hybrid populations between the two varieties. This information also reveals patterns of gene flow and population locations that have the highest conservation priority, which can be incorporated into efforts to conserve and protect this endangered species.