Abstract Mating systems and long-term climatic fluctuations have large impact on genetic diversity and population structure of plants; however, the genetic consequences have been poorly documented in species with geographic variation in mating systems. Here we used genetic variation from microsatellite markers to investigate the biogeographic pattern of Pachycereus pringlei , a columnar cactus with exceptional assortment of mating systems and wide range of distribution in the Sonoran Desert. Bayesian and multivariate analyses distinguished five genetic populations associated with differences in mating systems. Two hermaphroditic populations restricted to Cerralvo and Catalana islands were highly differentiated (Φ PT = 0.26 and 0.21, respectively). Cerralvo Island showed the lowest genetic diversity (H O = 0.26). Two other populations showed wide geographical distribution: a mostly gynodioecious population in the North comprising mostly hermaphrodite and female individuals, and a trioecious population in the South comprising unisexual and hermaphrodite individuals. The latter population exhibited the lowest level of genetic differentiation (Φ PT = 0.14) and highest genetic diversity (H O = 0.45). Cabo San Lucas, another trioecious population restricted to the southern end of the Baja California Peninsula, exhibits prevalence of unisexual individuals (0.76) but highly differentiated (Φ PT = 0.19) and relatively moderate genetic diversity (H O = 0.38). Latitudinal decrease of genetic diversity and isolation by distance supports northwards range expansion and southern BCP refugia, while Ecological Niche Models supported suitable climatic conditions during the Last Glacial Maximum in this region. These results suggest that the current biogeographic pattern of P. pringlei has been driven by longstanding climatic fluctuations associated with differential colonization abilities of genders and geographic variation of selfing and outcrossing rates. In conclusion, P. pringlei is not a panmictic assemblage and the interaction of historical vicariance and dispersal processes with mating systems may well explain present-day patterns of genetic variation.