Karyotypic and allozyme analyses were carried out on 55 individuals of Microtus subterraneus and M. majori from 19 populations in Europe and Anatolia. The occurrence of M. subterraneus in Asia Minor was proven. Standard karyotypes were found in M. majori (2n=54), M. subterraneus from Poland (2n=54), and central and south-eastern Europe (2n=52). Although the karyotype of M. subterraneus populations from Anatolia (2n=54), resembled that from Poland, there were apparent differences in the X chromosomes: it was larger and submetacentric (instead of metacentric) in the Anatolian populations owing to the presence of a large block of heterochromatin. The heterochromatic segment was also found to cause the size and shape differences in the X chromosomes between M. majori and European M. subterraneus (both in the 2n=52 and 2n=54 karyotypes). However, the location of this segment differed between the latter species and M. subterraneus from Anatolia. The distribution of the 2n=52 and both the 2n=54 karyotypes of M. subterraneus suggests that the Robertsonian fusion, distinguishing these different karyotypes, originated within the range of the species (i.e. within the 54-chromosome karyotype) and has spread subsequently towards its periphery. It is also suggested that both the fusion in European populations and amplification of heterochromatin in Anatolian populations are quite recent phenomena. The allozyme data indicate that gene flow is limited between populations of M. subterraneus and they are concordant with the isolation-by-distance model. There seems to be little correspondence between patterns of chromosomal and allozyme variation in M. subterraneus. Nei's genetic distance between M. subterraneus and M. majori was low (D=0.169), suggesting their recent divergence.