The genetic structure of Silurus glanis (Europe’s largest freshwater fish species) across most of its natural distribution was investigated using 10 microsatellite loci. The revealed levels of genetic diversity were much higher than previous allozyme and restriction fragment length polymorphism mitochondrial DNA analyses had shown; relative levels of variability among populations were however, in good agreement with the previous studies. Populations from large basins (Volga and Danube rivers) were the most polymorphic, while samples from the smaller Greek rivers, which are more prone to genetic bottleneck, exhibited the lowest levels of genetic diversity. Microsatellite multilocus genotyping permitted the assignment of individual fish to their population of origin with a score as high as 98.3%. Despite the great genetic differentiation of S. glanis populations, no consistent pattern of geographical structuring was revealed, in contrast to previous studies of European freshwater fish species. A model of isolation by distance seems more probable and a hypothesis of recent dispersion from only one glacial refugium is proposed. The discovery of the highest levels of microsatellite and mitochondrial diversity in the Volga sample and the presence of river connections, during the Pleistocene, between this area and all major areas of the present catfish distribution, place this refugium around the Ponto-Caspian region. Combining these data with those from previous studies, a number of markers are now available to monitor wild and hatchery populations even at the individual level.