There are several genetic studies of the grayling (Thymallus thymallus) in Central Europe, however, the phylogeography and genetic structure of the populations from some areas are still poorly recognized. The aim of this study was to determine the distribution of genetic diversity and the history of the postglacial migration of the species in Poland. Biological samples were collected in seven rivers and one fish farm. All specimens were genotyped at 12 microsatellite loci. Moreover, the sequence of ND5 and control region mtDNA gene was determined. We found clear differences in genetic diversity among the investigated population, with the lowest values found for the fish farm (Rutki) and intensively exploited river (Płytnica), and the highest for coastal and submontane rivers (Vistula, Wisłok, San). Phylogenetic analysis based on mtDNA suggests the presence of two postglacial colonization waves of grayling in Poland: (I) from the refugium of the Danube; (II) the Atlantic Basin. Currently, managing the populations on the European scale should involve stocking populations with the materials obtained from fish caught in the same rivers taking into account both environmental factors and rational angling. Stocking plans should consider local populations whose mixing will reduce the heterozygosity of the population without affecting the genetic pool.