Approaches using microsatellite markers are considered the gold standard for modern population-genetic studies. However, though they have found application in research into various platyhelminth taxa, they remained substantially underutilized in the study of monogeneans. In the present study, a newly-developed set of 24 microsatellite markers was used to investigate the genetic diversity of the generalist monogenean species D. vistulae. The analyzed parasite specimens were collected from 13 cyprinoid species from 11 sites in the Apennine and Balkan peninsulas. A total of 159 specimens were genotyped at each of the loci and the number of alleles per locus ranged from 2 to 16, with a mean number of 6.958 alleles per locus. Exceptionally high genetic diversity was observed among D. vistulae individuals in the southern Balkans, suggesting that this region might represent the center of diversification of the genus in Europe, from where Dactylogyrus parasites expanded into the north. The initial clustering analysis divided all investigated specimens into three major clusters; however, the results of the subsequent analyses revealed the existence of various subpopulations, suggesting that the population structure of D. vistulae is associated with the diversification of their cyprinoid hosts. In addition, partition of the parasite population was observed in regions of the sympatric occurrence of two host species, indicating that these hosts may represent a barrier to gene flow, even for generalist parasite species.