The underground vole Microtus thomasi, a Balkan endemic, displays remarkable variability in sex chromosome size and morphology. In the present study, we demonstrate this variability in two of its chromosomal races with 2n = 44 (i.e. 'thomasi' and 'atticus') with the use of C-banding on a sample of 189 individuals from 50 localities of Greece. In 'thomasi', five different, acrocentric X chromosome variants (X0-X4) are described, which differ significantly in size, due to heterochromatin addition. Also, three Y chromosome variants are described (Y0-Y2), ranging in size from very small (Y0) to large (Y2). The 'atticus' race displays three subtelocentric variants of the X chromosome (Xst0-Xst2), which differ in arm length ratio and heterochromatin content. In Peloponnesus, males of this race exhibit Y0 and Y1, whereas, in Attiki (south-east Sterea Ellada), males carry the small metacentric, Ym. Overall, there is a trend towards sex chromosome size increase in a south to north direction. We propose that the last glaciation must have restricted M. thomasi to a refugium in southern Peloponnesus. During post-glacial colonization, limited northward expansion of its distribution area must have been accompanied by consecutive heterochromatin addition, which is proven today by comparatively larger sex chromosomes in the northern populations of 'thomasi' and 'atticus' in Greece than in their southern populations.