The anuran species group Physalaemus gracilis comprises six species, and variation in the location of nucleolus organizer regions (NORs) was observed across the four species that have been karyotyped to date. The NORs are located interstitially on chromosome 8 of P. carrizorum Cardozo et Pereyra, 2018, and P. lisei Braun et Braun, 1977, terminally on chromosome 8 of P. gracilis (Boulenger, 1883), and terminally on chromosome 10 of P. barrioi Bokermann, 1967. To contribute to the comparative analysis of this group, including the assessment of the hypothesis of homology among these NOR-bearing chromosomes, we described the karyotype of P. evangelistai Bokermann, 1967, and expanded the cytogenetic analyses of P. carrizorum, P. lisei, and P. barrioi. We used classical cytogenetic techniques and mapped, by fluorescent in situ hybridization (FISH), two repetitive sequences: the PcP190 satellite DNA and the U2 snRNA gene. Physalaemus evangelistai exhibited a 2n = 22 karyotype, with meta- and submetacentric chromosomes, which corresponds to the typical karyotypic configuration of the genus. We found an interstitial heterochromatin DAPI-positive band on the short arm of the NOR-bearing chromosomes 8 of P. evangelistai and P. carrizorum from Palmas-PR, and chromosome 10 of P. barrioi, which corroborates the hypothesis that these chromosomes are homologous. In P. evangelistai, an additional NOR was observed on chromosome 9 of females. Moreover, the karyotype of P. carrizorum from Palmas-PR differed from that previously described for P. carrizorum from Misiones, particularly in the number of PcP190 clusters and intrachromosomal position of the NOR on chromosome 8. Specimens from Palmas-PR showed a terminal NOR on chromosome 8 and PcP190 clusters on chromosomes 1 and 3, whereas those from Misiones had an interstitial/pericentromeric NOR on chromosome 8 and a single PcP190 cluster on chromosome 3. Further analyses are still needed to assess whether these cytogenetic differences represent interspecific variation.