We report the first karyotypic descriptions of Nesomyinae, a subfamily of rodents endemic to Madagascar. Using standard staining as well as G-banding and C-banding we detected karyotypic variation at the intergeneric, interspecific and intraspecific levels among six specimens referable to the species Eliurus majori, Eliurus minor, Eliurus tanala and Nesomys rufus. The two E. minor specimens analysed (2n = 74 and 76) differ from the two E. tanala specimens (2n = 74 and 75) by a minimum of 15 pericentric inversions (or centromeric shifts) suggesting that karyotypic orthoselection may be canalizing rearrangements in this species. In turn, the karyotype of E. minor appears to differ from E. majori (2n = 58) and N. rufus (2n = 60) by a series of more complex rearrangements involving multiple pericentric inversions (or centromeric shifts) and Robertsonian translocations. We discuss the presence of karyotypic variation in these nesomyine species in light of some environmental constraints that are known to have driven the evolution of the Malagasy vertebrate fauna.