To examine the within- and among-population genetic structure of Plamopara viticola oosporic populations in Europe, 8991 lesions from 32 vineyard plots were collected and analysed. Four multi-allelic microsatellite markers were used to genotype the pathogen. All populations had high levels of gene and genotypic diversity. Most populations were in Hardy–Weinberg equilibrium and thus randomly mating. Among P. viticola populations, significant low to moderate genetic differentiation was observed, even between geographically close populations. This genetic differentiation was also evident in the neighbour-joining phylogenetic genetic distance tree, showing clear substructure and distinguishing mainly five clusters based on geographical origin. Significant isolation by distance was found in central European P. viticola populations, suggesting a step-wise migration model. No significant isolation by distance was found within Greek populations, most probably owing to natural geographical barriers such as the sea and mountains, as well as the frequent population bottlenecks occurring in these populations, preventing natural migration among populations. The high variability of P. viticola provides explanation for its successful infestation of the heterogeneous European vineyards in the last 125 years after its introduction.