The butterfly Proclossiana eunomia (Lepidoptera: Nymphalidae) was discovered at a single locality in the Czech Republic in 1963. Until the 1980s, it was known from a restricted area, from which it spontaneously expanded during the 1980s to other localities up to 23 km from the source population. Samples were collected in 2002 from the source and ten other populations, totalling 274 specimens. All samples were analysed by electrophoresis for four polymorphic loci. Mean heterozygosity decreased with distance from the source population; this suggested a process of stepping stone colonization, involving the loss of rare alleles along the way. The populations close to the source population (less then ca. 15 km) retain a similar heterozygosity, whereas populations further away have a much reduced heterozygosity. Such a pattern of genetic differentiation and founder effect within a region is typical of specialized species with relatively low dispersal ability. The high level of genetic polymorphism found in the Šumava populations suggests that populations of this northern species in temperate-zone mountains are not just outposts of otherwise huge northern distribution, but represent genuine phylogeographic refugia. Survival of such species depends on the survival of the source population and of a sufficiently dense network of habitat patches.