Background: The blind mole rats (Spalacinae) are fossorial rodents exhibiting exceptionally extensive variation in the karyotype. The taxonomy of this group is not definitively resolved, and the species discrimination is often not clear, even with the use of chromosomal analyses. Results: We have studied the karyotype of three populations of blind mole rats (Spalacinae) from Anatolia classified tentatively as Nannospalax xanthodon (Bolu and Içel provinces) and Nannospalax ehrenbergi (Gaziantep province). C-banding and AgNOR staining were used in the cytogenetic analysis. In all populations, the karyotype comprised 52 chromosomes including 8 to 10 bi-armed and 17 to 15 acrocentric autosomal pairs. The centromeric position varied in the sex chromosomes between the populations. The C-positive centromeric bands occurred in most of the bi-armed autosomes and the sex chromosomes and in some of the acrocentric autosomes. The nucleolar organizer regions were localized in three (N. xanthodon) or five (N. ehrenbergi) autosomal pairs. Conclusions: It is difficult to find particular chromosomal differences between the studied populations which could enable unequivocal species identification. This finding emphasizes the need of taxonomic revision of the species structure within the Nannospalax genus.