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Aim Climatic changes and fluctuations in the past have strongly influenced the distribution of animal and plant species. Such fluctuations are also reflected in the patterns of genetic diversity on both local and global scales. The genetic pattern of the pearly heath butterfly, Coenonympha arcania, was used to evaluate the genetic differentiation of isolated (in north-western Europe), peripheral (in north-eastern Europe) and central (in southern Europe) populations in the context of post-glacial distributional changes of the species. Location Europe (Sweden, Germany, the Baltic states, Italy, Slovenia, Hungary, Romania, Bulgaria). Thus, samples were collected from large parts of the species’ distribution representing the three categories mentioned above. Methods We analysed 18 loci of 569 individuals from 28 populations by allozyme electrophoresis. We used both individual-based and population-based analyses, including F-statistics, various clustering methods and Markov chain Monte Carlo simulations. Results All loci, except Fum, were polymorphic. The mean FST for all samples was 0.18. The mean genetic distance among populations was 0.046. Two major genetic lineages were distinguished. Populations from the centre of the distributional range in southern Europe and the northern periphery of the distributional range differed significantly in their level of genetic variability. The central populations of south-eastern Europe showed high levels of genetic diversity and no differentiation among populations. Main conclusions Most probably the two major genetic lineages evolved during glacial isolation in two disjunct Mediterranean refugia. The lack of genetic differentiation across south-eastern Europe implies a continuous Würm ice age distribution in this area, thus supporting the functional existence of steppe forests throughout this region. The peripheral-isolated populations in Sweden seem to have suffered from one or more severe bottlenecks, resulting in substantial genetic impoverishment. The peripheral-connected eastern Baltic populations, on the other hand, are affected by post-glacial and possibly recurrent gene flow from more central parts of the distribution. [ABSTRACT FROM AUTHOR]