Using cross-species chromosome painting, we have carried out a comprehensive comparison of the karyotypes of two Ellobius species with unusual sex determination systems: the Transcaucasian mole vole, Ellobius lutescens (2n = 17, X in both sexes), and the northern mole vole, Ellobius talpinus (2n = 54, XX in both sexes). Both Ellobius species have highly rearranged karyotypes. The chromosomal paints from the field vole (Microtus agrestis) detected, in total, 34 and 32 homologous autosomal regions in E. lutescens and E. talpinus karyotypes, respectively. No difference in hybridization pattern of the X paint (as well as Y paint) probes on male and female chromosomes was discovered. The set of golden hamster (Mesocricetus auratus) chromosomal painting probes revealed 44 and 43 homologous autosomal regions in E. lutescens and E. talpinus karyotypes, respectively. A comparative chromosome map was established based on the results of cross-species chromosome painting and a hypothetical ancestral Ellobius karyotype was reconstructed. A considerable number of rearrangements were detected; 31 and 7 fusion/fission rearrangements differentiated the karyotypes of E. lutescens and E. talpinus from the ancestral Ellobius karyotype. It seems that inversions have played a minor role in the genome evolution of these Ellobius species.