The range of Phyllomedusa rohdei was recently extended to the state of Bahia, Brazil. Cytogenetic analysis through conventional Giemsa staining and Ag-NOR banding of P. rohdei samples from two localities in the Atlantic Rainforest, state of Bahia revealed intraspecific karyotype variation. Although all individuals showed the diploid number of 2n  =  26, variations in the karyotypic formula were observed. The greatest variation was found in the length of first chromosome pair. The Ag-NOR banding identified variability in the number of stained chromosomes, which ranged from two to four banded chromosomes per individual. The results revealed the preliminary stages of chromosomal differentiation within this species.