Karyotypic polymorphism of five taxa of the rodent genus Rhipidomys from the Brazilian Amazon and Cerrado biomes was analysed. Rhipidomys nitela Thomas, 1901 from Amazon has 2n = 48, FN = 68. The other species, all have 2n = 44, but can be separated into two groups, one with high FNs (76, 80) and the other with low FNs (48, 52). Two cytotypes of R. mastacalis (Lund, 1840) with high FNs were trapped in four localities of the Cerrado, showing 19 and 17 biarmed autosomes, respectively. A low FN (48) was observed in R. leucodactylus (Tschudi, 1844) in two localities of the Cerrado and FN = 52 in one locality in the Cerrado and the Amazon. All taxa with 2n = 44 have a medium-sized acrocentric X chromosome and a small Y. Rhipidomys nitela is different from the species with 2n = 44 by presenting a heterochromatic short arm of the X chromosome. In all karyotypes analysed, the nucleolus organizer regions were located in the short arms of two to six pairs and the (T2AG3)n telomeric probes hybridized in situ in both the short and long arms of all pairs of the karyotypes.