We studied populations of Stenocereus pruinosus throughout Mexico, a species important for its edible fruit. The Tehuacán Valley is setting of ancient and the currently most active management of S. pruinosus; we hypothesized Tehuacán as the original area of domestication of S. pruinosus and expected to find there its highest genetic variation and original source of genes of cultivated plants. Through nuclear microsatellite loci we studied spatial distribution of genetic variation and population differentiation. We sampled wild and managed populations of the Central-western (Tehuacán, Central Valleys and Tehuantepec, Oaxaca), north-eastern (Huasteca) and south-eastern (Chiapas) regions. Differences among regions and populations were compared through homogeneity and exact test for FIS , AMOVA, Bayesian analysis, and genetic barriers. A niche analysis allowed corroborating taxonomic identity of populations. The highest genetic diversity was in Tehuantepec (HE = 0.841), decreasing towards the extremes of distribution (HE = 0.242 in Huasteca, HE = 0.254 in Chiapas). Genetic structure is significantly high among populations and regional groups, differentiating one group formed by northern and southern populations and other formed by populations of the Central-western region. Differences among groups suggested that populations from Huasteca could be species different to S. pruinosus, but the niche analysis did not support such hypothesis. Populations from Tehuantepec were different but genetically interconnected with those of Tehuacán. Tehuantepec is the main reservoir of genetic diversity of wild populations of S. pruinosus, but Tehuacán is the principal current area of domestication of S. pruinosus and probably where its domestication originated. Conclusions would be stronger by analyzing DNAc lineages.