Plasmopara halstedii is a biotrophic parasite of sunflower (Helianthus annuus). We genetically analysed 162 P. halstedii samples collected between 1982 and 2018, representing a 3500 km west-east transect across Europe, from France to the Krasnodar region in southern Russia. To assess the population genetic structure among P. halstedii isolates, we sequenced two mitochondrial regions (cox1 and cox2), in addition to analysing eight polymorphic simple sequence repeat (SSR, microsatellite) markers.Sequencing of cox2 enabled comparison of our data with recent SSR-based studies to provide further evidence for differences among P. halstedii strains infecting different hosts (H. annuus, H. × laetiflorus, Rudbeckia spp.). There are two different lineages infecting H. annuus that we resolved on a neighbour-joining tree, which also corresponded to a discriminant analysis of principal components. This suggests at least two independent introductions of P. halstedii on sunflower. We observed no relationship between the phylogenetic placement of the individual samples and virulence characteristics, indicating independent evolution of virulence phenotypes. Both genetic markers clearly separated P. halstedii samples originating from H. annuus and H. × laetiflorus, suggesting these might represent distinct species.