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The population structure of Hymenoscyphus fraxineus, the causal agent of ash dieback, was assessed at four closely located sites in the Czech Republic. To analyse the genetic variation, one Swiss and one Norwegian population with known population structures were selected as reference points. The analysis was performed using 16 previously published and five newly developed microsatellite markers. The quality of the new markers was assessed by sequencing the flanking region, identifying the type of the mutation and analysing the inheritance and linkage between all pairs of loci. In addition, markers were tested on 45 Japanese strains to confirm their usability on native H. fraxineus populations in Asia. One of the new markers was monomorphic in all European populations and one marker exhibited a high percentage of null alleles in the Japanese samples. Twenty markers in the four Czech populations showed lower average gene diversity than in the other two European populations. One Czech population significantly differed from all the others, with a pairwise GST of approximately 0.2, the lowest allelic richness and very low average gene diversity, likely resulting from a founder effect. The amount of genetic differentiation between the four neighbouring Czech populations exceeds the expected value from previous Europe-wide population studies and suggests that local population dynamics can affect the population structure of H. fraxineus. [ABSTRACT FROM AUTHOR]