Abstract: The Neotropical ants Pachycondyla crenata (Roger, 1861) and Pachycondyla mesonotalis (Santschi, 1923) (Hymenoptera: Formicidae: Ponerinae) nest in pre-existing hollow cavities on trees in native and planted forests where they form discrete colonies. Their morphology is very similar; only their body size appears different. They are sympatric in Brazil, but P. crenata can be found further north of the Neotropical Region than P. mesonotalis. Aiming to evaluate the character differentiation in these taxa, cytogenetic studies were carried out and complemented by morphometric observations. Cytogenetic preparations were made using prepupae gathered from two Brazilian sites. Both taxa have a diploid karyotype composed of 26 chromosomes. The karyotype formula for P. crenata is 2K = 2M + 24A, while for P. mesonotalis it is 2K = 26A. A pericentric inversion Am on a single pair of chromosomes may have given rise to the difference between the species. Although it is not possible to pinpoint the more ancestral karyotype between P. crenata and P. mesonotalis, we speculate that the reported chromosome rearrangement was the differentiation mechanism that prompted their species divergence, resulting only in morphometric differences with a minimum impact on the ecology and morphology, yet allowing them to live sympatrically.