The genus Chelus, commonly known as Matamata is one of the most emblematic and remarkable species among the Neotropical chelids. It is an Amazonian species with an extensive distribution throughout Negro/Orinoco and Amazonas River basins. Currently, two species are formally recognized: Chelus orinocensis and Chelus fimbriata and although it is still classified as "Least Concern" in the IUCN, the Matamatas are very appreciated and illegally sold in the international pet trade. Regardless, little is known regarding many aspects of its natural history. Chromosomal features for Chelus, for instance, are meagre and practically restricted to the description of the diploid number (2n = 50) for Chelus fimbriata, and its sex determining strategies are yet to be fully investigated. Here, we examined the karyotype of Chelus fimbriata and the newly described Chelus orinocensis, applying an extensive conventional and molecular cytogenetic approach. This allowed us to identify a genetic sex determining mechanism with a micro XY sex chromosome system in both species, a system that was likely present in their most common recent ancestor Chelus colombiana. Furthermore, the XY system found in Chelus orinocensis and Chelus fimbriata, as seen in other chelid species, recruited several repeat motifs, possibly prior to the split of South America and Australasian lineages, indicating that such system indeed dates back to the earliest lineages of Chelid species.