Tropical fruit trees are an important component of the human diet; however, little is known about their genetic diversity levels. Zapote mamey (Pouteria sapota) is a tree native to southeastern Mexico and Central America, and Mexico is the leading producer in the world. Studies of the genetic diversity of Zapote mamey have been based on cultivated materials using morphological and biochemical characterization or dominant molecular markers. To gain a deeper understanding about the conservation status of Zapote mamey in its center of origin and domestication, we collected 188 individuals from eight wild and five cultivated populations in southeastern Mexico and characterized them using eight microsatellite loci. STRUCTURE, 3D-PCoA, and neighbor-joining analyses showed three groups in the wild gene pool and one group in the cultivated gene pool. F ST values were significant between wild and cultivated gene pools, among the four groups observed and among the 13 populations collected (0.13, 0.25, and 0.36, respectively). Overall, we found low levels of genetic diversity (A = 2.77, H O = 0.29, H E = 0.39), permutation tests did not show significant differences between wild and cultivated gene pools. The Garza-Williamson index showed low values in both gene pools (wild = 0.16, cultivated = 0.11) and the Bottleneck program indicated a decrease in genetic diversity in both gene pools (wild, P = 0.027; cultivated, P = 0.054); both analyses suggest a potential genetic bottleneck within this species. This study can help to generate adequate sampling techniques and to develop effective management strategies for Zapote mamey of southeastern Mexico.