Abstract Boana comprises a diverse genus of Neotropical treefrogs, currently rearranged into seven taxonomic species groups. Although cytogenetic studies have demonstrated diversity in its representatives, the chromosomal mapping of repetitive DNA sequences is still scarce. In this study, Boana albopunctata, Boana faber, and Boana prasina were subjected to in situ localization of different repetitive DNA units to evaluate trends of chromosomal evolution in this genus. Boana faber and B. prasina had 2n=24 chromosomes, while B. albopunctata has 2n=22 and an intra-individual variation related to the presence/absence of one B chromosome. The location of 45S rDNA sites was different in the analyzed karyotypes, corroborating with what was found in the distinct phylogenetic groups of Boana. We presented the first description of 5S rDNA in a Boana species, which showed markings resulting from transposition/translocation mechanisms. In situ localization of microsatellite loci proved to be a helpful marker for karyotype comparison in Boana, commonly with cis accumulation in the heterochromatin. On the other hand, genomic dispersion of microsatellites may be associated with hitchhiking effects during the spreading of transposable elements. The obtained results corroborated the independent diversification of these lineages of species from three distinct phylogenetic groups of Boana.