We performed a comparative analysis of the nucleolus organizer region (NOR) distribution in the karyotypes of hylid frogs from two different taxonomic groups, Hypsiboas faber and H. semilineatus. Silver nitrate staining of NORs (Ag-NORs) and fluorescence in situ hybridization (FISH) with a rDNA probe were used to investigate the chromosomal location of rDNA loci in two species. The karyotype of H. semilineatus and the Ag-NORs distribution of the four species are presented herein for the first time. After conventional staining, the four species presented very similar karyotypes with 2n = 24, but Ag-NORs analyses revealed species-specific characteristics. H. albomarginatus, H. faber and H. semilineatus had one pair of interstitial Ag-NORs in the short arm of pairs 2, 11, and in the long arm of pair 7, respectively. H. pardalis presented telomeric NORs in the long arm of pair 11. Ag-NORs were heteromorphic in three of the species (H. pardalis, H. semilineatus and H. albomarginatus) and FISH confirmed the differential activity of rDNA genes in H. semilineatus. In the present study, 2n = 24 karyotypes could be distinguished by their Ag-NORs distribution. Our results further the knowledge about the cytogenetics of hylids from Brazil.