BACKGROUND Rhipidomys is the second most specious and the most widespread genus of the tribe Thomasomyini. Chromosomal data have been an important tool in the taxonomy of the group that presents low variability of diploid number (2n) and highly variable fundamental numbers (FN). Despite such diversity, the genus has been studied mainly by classical and banding cytogenetic techniques. METHODS This study performed a comparative study between R. emiliae (2n = 44, FN = 52), R. macrurus (2n = 44, FN = 49), R. nitela (2n = 50, FN = 71), and R. mastacalis (2n = 44, FN = 72) using chromosome painting probes of two Oryzomyini species. RESULTS Our analysis revealed pericentric inversion as the main rearrangement involved in the karyotype evolution of the group, although tandem fusions/fissions were also detected. In addition, we detected eight syntenic associations exclusive of the genus Rhipidomys, and three syntenic associations shared between species of the tribe Thomasomyini and Oryzomyini. CONCLUSIONS Comparative cytogenetic analysis by ZOO-FISH on genus Rhipidomys supports a pattern of chromosomal rearrangement already suggested by comparative G-banding. However, the results suggest that karyotype variability in the genus could also involve the occurrence of an Evolutionary New Centromere.