Mole rats (genus Nannospalax) display prolific chromosomal variation with more than 50 distinct cytotypes. These cytotypes are largely indistinguishable morphologically, are mainly allopatric and their taxonomic ranking is contradictory. We established a cytochrome b phylogeny for 15 cytotypes belonging to all three species recognized on morphological grounds (morphospecies): N. leucodon, N. xanthodon and N. ehrenbergi. Phylogenetic reconstructions yielded two highly divergent groups which are in agreement with the current division into two subgenera (Nannospalax and Mesospalax). The former comprised samples from south-eastern Turkey, Israel and Egypt (the morphospecies N. ehrenbergi). Basal dichotomy within Mesospalax remained unresolved and the putative sister position of N. leucodon against the two lineages of N. xanthodon was not supported in our analysis. Net divergences between sister cytotypes were low (< 2.0%) and two N. leucodon cytotypes were not even reciprocally monophyletic. Among the three morphospecies, the genetic diversity was lowest in N. leucodon (2.4% ± 0.3%), highest in N. xanthodon (8.8% ± 0.7%) and intermediate in N. ehrenbergi (5.0% ± 0.5%). Our results show that associations between genetic and chromosomal variation are not widespread and common in mole rats, and therefore refute the generalization of a ‘cytotype-equals-species’ approach. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105, 446–455.