The Virginia opossum (Didelphis virginiana) is considered highly adaptable to anthropogenic disturbances; however, the genetic effects of disturbance on this marsupial have not been studied in wild populations in Mexico. Here we evaluated the genetic diversity of D. virginiana at sites with different levels of disturbance within the Highlands and Central Depression regions of Chiapas in southern Mexico. Twelve microsatellite loci were used and the results demonstrated moderate mean heterozygosity (He = 0.60; Ho = 0.50). No significant differences in heterozygosity were found among sites with different levels of disturbance in both regions (range Ho = 0.42–0.57). We observed low but significant levels of genetic differentiation according to disturbance level. The inbreeding coefficient did not differ significantly from zero, suggesting that low genetic differentiation in these environments may be associated with sufficient random mating and gene flow, a result associated with the high dispersal and tolerance characteristics of this marsupial. Our results for D. virginiana in this particular area of Mexico provide a foundation for exploring the impact of human disturbance on the genetic diversity of a common and generalist species.