We determined the overall karyotypic structure, positions of NORs, as well as the distribution of constitutive heterochromatin for Epipedobates flavopictus, E. trivittatus, E. femoralis, and E. hahneli. Despite a conserved chromosome number (2N = 24), morphological differences were seen in the group of small chromosomes. Epipedobates femoralis presented a distinctive karyotype compared to the other species analyzed. All species examined had NORs on different chromosomes. The C-banding pattern showed a considerable variation among the species. Epipedobates flavopictus is remarkably different from the others in possessing only centromeric C-bands. The distribution of heterochromatin varied among species and seems to evolve as a species-specific trait. We suggest that NOR location variability indicates that some rearrangement mechanisms have taken place during the evolutionary history of this group, because Dendrobatidae is considered a monophyletic taxon. We do not discard that some general pattern in either C-band or NOR location, or both, may emerge when more species of this genus are analyzed.