The genus Engystomops is divided into two groups, namely the Duovox clade and the Edentulus clade. The species of Edentulus clade have karyotypes with 2n = 22, while E. pustulatus and E. puyango, which belong to Duovox clade, have 2n = 20. To investigate if 2n = 20 is a synapomorphy of Duovox clade, we cytogenetically analyzed all the species of this group, except for E. puyango, in the present study. All of them had 2n = 20, differing from the species of Edentulus clade. Since the species already karyotyped of the genus Physalaemus, which is considered to be the sister group of Engystomops, also have 2n = 22, we conclude that the 2n reduction is a synapomorphy of Duovox clade. Despite the karyotypes of all the species of Duovox clade were very similar, they varied in the NOR pattern. In E. coloradorum, an additional NOR was found in one homologue of the chromosome pair 10 exclusively in all females, indicating that this could possibly be a sexual pair of the ZZ/ZW system. Also in this species, it was found the first case of natural polyploidy of the genus Engystomops.