Crop wild relatives represent an important agronomic resource for crop improvement and biodiversity conservation. The wild squash Cucurbita argyrosperma subsp. sororia. (Cucurbitaceae) has been considered the wild ancestor of cultivated forms of C. argyrosperma. In order to characterise the geographic patterns of genetic variation in this wild cucurbit and to identify priority areas for conservation, we analysed the genetic diversity and structure of natural populations along the Mexican Pacific coast. By using 14 polymorphic microsatellites, we genotyped 378 individuals sampled from 61 locations. Standard population genetics analyses and group testing were conducted on the genotypes with the aid of principal coordinate analysis and Bayesian analysis. Overall, we found an average of 12.3 alleles per locus and an expected heterozygosity of 0.756. We found greater genetic diversity in southern populations. The fixation index was 0.113, suggesting a mixed mating system. The Mantel test revealed a minor distance effect on genetic differentiation between individuals (r = 0.321). Finally, we found three main groups of populations arranged in a mostly latitudinal pattern, from Sinaloa (north-west) to Oaxaca–Guerrero (south-east). The greater genetic diversity and heterogeneity among southern populations (Guerrero–Oaxaca), suggests that this region is an important centre of diversity of this wild squash with important implications for conservation.