To the Neotropical genus Acrocomia (Arecaceae) is attributed eight species with a wide distribution in America. A. aculeata and A. totai are the most important species because of their high economic potential for oil production. However, there is no consensus in their classification as different taxons and their distinctiveness is particularly challenging due to morphological similarities with large plasticity of the traits. In addition, there is doubt about the occurrence of interspecific hybrids between both species. In this study, we applied a genetic population approach to assessing the genetic boundaries, diversity and to identify interspecific hybrids of A. aculeata and A. totai. Thirteen loci of simple sequence repeat (SSR) were employed to analyze twelve populations representing a wide distribution of species, from Minas Gerais, Brazil to Formosa, Argentina. Based on the Bayesian analysis (STRUCTURE and NewHybrids) and Discriminant Analysis of Principal Components (DAPC), our study supports the recognition of A. aculeata and A. totai as two species and the estimates of genetic parameters revealed more genetic diversity in A. totai (H_E=0.551) than in A. aculeata (H_E=0.466). We obtained evidence of hybridization between the species and that admixed individuals were assigned as F2 hybrids. In conclusion, this study showed the usefulness of microsatellite markers to elucidate the genetic boundaries of A. aculeata and A. totai, supporting their classification as different species and increase our knowledge about genetic diversity at the level of populations and species. The results are essentials to establish strategies for the adequate management, conservation, and domestication of both species.