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The genus Phyllomedusa has been the target of regular taxonomic investigations. The species Phyllomedusa nordestina was recently separated from P. hypochondrialis. Morphological variations in the P. rohdei interpopulation have already been reported, suggesting the existence of more than one taxon under that name. In the present study, we have cytogenetically characterized two populations of P. nordestina and one of P. rohdei. Both species displayed 2n = 26 chromosomes with 12 metacentric, 12 submetacentric and 2 subtelocentric chromosomes. The C‐banding analyses revealed discrete differences in the quantity of centromeric heterochromatin between the two species. The nucleolus organizer region (NOR) was detected in pair 9 of both species, but is located in the pericentromeric region of the short arm in P. nordestina and in the long arm subtelomeric region of P. rohdei. Chromosomal data from this …