The Balkan endemic species Microtus (Terricola) thomasi – Thomas’ vole – is widely distributed in the Greek mainland and presents regionally confined karyotypic forms. In order to test the significance of geographical isolation and/or karyological differences in microevolutionary processes at the intraspecific level we used restriction enzymes and partial sequencing to study the polymorphism of the mtDNA d-loop control region in ten Greek populations. An overall high rate of both inter- and intrapopulation polymorphism was observed. The mtDNA diversity was unevenly distributed so that most of the populations studied were very similar and closely related, and only a few of them were clearly differentiated from the others. The mtDNA analysis failed to reveal any clear variation pattern that could be correlated with karyotypic forms or geographical proximity of the populations studied. Instead, the mtDNA phylogeny gave evidence in support of the coexistence of non-polymorphic (2n = 44) and polymorphic (2n < 44) karyotypic forms within the ancestral Greek M. (T.) thomasi stock. Interestingly, only one population (Agios Stefanos) seemed to significantly diverge from all the others in both methods used, forming a well-supported paraphyletic clade. Allozymic studies have already reported similar results, corroborating the genetic peculiarity of this population. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 55–68.