Cycloramphus bolitoglossus (Werner, 1897) is a rare species with a low population density in the Serra do Mar region of Paraná and Santa Catarina, in southern Brazil. Currently, it has been assigned to the Near Threatened (NT) category in the Brazilian List of Endangered Animal Species. Here, we described the karyotype of this species for the first time and investigated the patterns of some repetitive DNA classes in the chromosomes using molecular cytogenetic approaches. We isolated, sequenced and mapped the 5S rDNA and the satellite DNA PcP190 of C. bolitoglossus, as well as mapped the telomeric sequences and seven microsatellites motifies [(GA).sub.15, (CA).sub.15, (GACA).sub.4, (GATA).sub.8, (CAG).sub.10, (CGC).sub.10, and (GAA)].sub.10 . Cycloramphus bolitoglossus has 2n = 26 chromosomes and a fundamental number (FN) equal to 52, with a highly conserved karyotype compared to other genus members. Comparative cytogenetic under the phylogenetic context of genus allowed evolutionary interpretations of the morphological changes in the homologs of pairs 1, 3, and 6 along with the evolutionary history of Cycloramphus. Two subtypes of 5S rDNA type II were isolated in C. bolitoglossus genome, and several comparative analysis suggests mixed effects of concerted and birth-and-death evolution acting in this repetitive DNA. The 5S rDNA II subtype "a" and "b" was mapped on chromosome 1. However, their different position along chromosome 1 provide an excellent chromosome marker for future studies. PcP190 satellite DNA, already reported for species of the families Hylidae, Hylodidae, Leptodactylidae, and Odontophrynidae, is scattered throughout the C. bolitoglossus genome, and even non-heterochromatic regions showed hybridization signals using the PcP190 probe. Molecular analysis suggests that PcP190 satellite DNA exhibit a high-level of homogenization of this sequence in the genome of C. bolitoglossus. The PcP190 satDNA from C. bolitoglossus represents a novel sequence group, compared to other anurans, based on its hypervariable region. Overall, the present data on repetitive DNA sequences showed pseudogenization evidence and corroborated the hypothesis of the emergence of satDNA from rDNA 5S clusters. These two arguments that reinforced the importance of the birth-and-death evolutionary model to explain 5S rDNA patterns found in anuran genomes.