Pseudoponera stigma (F.) and Pseudoponera gilberti (Kempf) (Hymenoptera: Formicidae) are closely related Neotropical ants, often misidentified due to their morphological similarities. These species also share behavioral and ecological characters. In this study, we examined cytogenetic approaches as a tool to aid identification of P. stigma and P. gilberti. Both numerical and morphological karyotypic variations were identified based on different cytogenetic techniques. The karyotype formula of P. stigma, 2K = 10M 4SM differs from that of P. gilberti, 2K = 10M 2SM, and the CMA3 /DAPI- sites also differ, allowing both species to be distinguished by chromosomal characters.