Physalaemus biligonigerus, P. fuscomaculatus and P. sp. have 2n=22 chromosomes, consistent with the number of chromosomal pairs found in the genus. There were no substantial differences in the karyotypes of the three species, except for chromosome 3 which was submetacentric in P. fuscomaculatus and metacentric in P. biligonigerus and P. sp. All chromosomes were characterised by the presence of a C-band in the pericentromeric region. Some interstitial bands were also detected at the same position in all karyotypes, but the band located on the short arm of chromosome 3 was larger in P. biligonigerus and P. sp. than in P. fuscomaculatus. Also, only P. sp. had a large block of pericentromeric heterochromatin in the short arm of chromosome 9 and another one in the telomeric band on the long arm. The NOR was located on the long arm of chromosome 9 in the three species. These findings confirmed that the three species are indeed closely related cytogenetically.5341732283288