The differentiation of sex chromosomes is thought to be interrupted by relatively frequent sex chromosome turnover and/or occasional recombination between sex chromosomes (fountain-of-youth model) in some vertebrate groups as fishes, amphibians, and lizards. As a result, we observe the prevalence of homomorphic sex chromosomes in these groups. Here, we provide evidence for the loss of sex chromosome heteromorphism in the Amazonian frogs of the genus Engystomops, which harbors an intriguing history of sex chromosome evolution. In this species complex composed of two named species, two confirmed unnamed species, and up to three unconfirmed species, highly divergent karyotypes are present, and heteromorphic X and Y chromosomes were previously found in two species. We describe the karyotype of a lineage estimated to be the sister of all remaining Amazonian Engystomops (named Engystomops sp.) and perform chromosome painting techniques using one probe for the Y chromosome and one probe for the non-centromeric heterochromatic bands of the X chromosome of E. freibergi to compare three Engystomops karyotypes. The Y probe detected the Y chromosomes of E. freibergi and E. petersi and one homolog of chromosome pair 11 of Engystomops sp., suggesting their common evolutionary origin. The X probe showed no interspecific hybridization, revealing that X chromosome heterochromatin is strongly divergent among the studied species. In the light of the phylogenetic relationships, our data suggest that sex chromosome heteromorphism may have occurred early in the evolution of the Amazonian Engystomops and have been lost in two unnamed but confirmed candidate species.