The genus Ctenomys comprises a group of rodents with remarkable karyotypic variability linked to the distribution of repetitive sequences and rearrangements. We analyzed the distribution and variation of repetitive DNA in two parapatric Andean species from Chile: Ctenomys maulinus brunneus (2n = 26; FNa = 48) and Ctenomys sp. (2n = 28; FNa = 50). Self-genomic in situ hybridization (Self-GISH), whole comparative genomic hybridization (W-CGH), and fluorescent in situ hybridization (FISH) using a telomeric probe were performed. Phylogenetic relationships and divergence times based on cytochrome b sequences helped infer the direction and timing of cytogenetic changes. Self-GISH revealed the absence of highly repetitive sequences in four chromosome pairs of C. m. brunneus and nine in Ctenomys sp. W-CGH showed no differential expansion of species-specific repeats, suggesting no recent major sequence turnover. FISH detected signals exclusively in telomeres. Phylogenetic analyses indicate that C. m. maulinus (2n = 26) diverged from the clade formed by C. m. brunneus and Ctenomys sp. during the Late Pleistocene, supporting, together with cytogenetic data, a loss of repetitive sequences associated with fission events from 2n = 26 to 28. These findings highlight the evolutionary significance of repetitive DNA and reinforce Ctenomys as a model for studying chromosomal evolution.