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Numerous repetitive DNA elements found in the huge frog genomes have contributed significantly to this vertebrate group's karyotypic diversity. The anurans in the family Pipidae are thought to have evolved mostly through intrachromosomal rearrangements based on their presumed primitive karyotype (2n=20). All other genera are located in Africa, with Pipa being the only South American pipid. At least 136 million years ago, the South American lineages split apart from the African ones, and it is believed that a significant biogeographic factor played a role in this process. Here, we explored the potential of repetitive DNA to enhance our comprehension of karyotype differentiation within the Pipidae family and to broaden our ability to decipher chromosomal evolution within this frog family. Our findings support hypotheses on the chromosomal homologies between the species in pairings 6, 8, and 9 by demonstrating a lengthy history of conservation in the chromosome containing the H3 histone locus. The chromosomal distribution of the microsatellite motifs also offers helpful markers for comparative genomics between Pipa carvalhoi and Xenopus tropicalis at the chromosome level, providing fresh perspectives on how the karyotypes of these species have evolved. We found patterns in the distribution and abundance of the microsatellite arrangements that were similar, reflecting the shared chromosomal organisation between these two species in the terminal/subterminal region. The longitudinal differentiation of pairs that are identical in size and form, such as pairs 1, 2, 4, and 5, was made possible by the microsatellite probes, which discovered a different arrangement of the repetitive DNA among the chromosomes of the two species. With a clear enrichment of the CA and GA microsatellite motifs in P. carvalhoi, we also discovered evidence of the different composition of the repeating motifs of the centromeric region between the species investigated in the current study. Last but not least, Interstitial Telomeric Sequences (ITS) and microsatellite enrichment in the pericentromeric region of chromosome pairs 6, 8, and 9 in the P. carvalhoi karyotype support the idea that pericentromeric inversions happened during the chromosomal evolution of P. carvalhoi and support the role of repetitive DNA in the remodelling of the karyotype architecture of the Pipida
