Assortative mating resulting from host plant specialization has been proposed to facilitate rapid ecological divergence in biotrophic plant pathogens. Downy mildews, a major group of biotrophic oomycetes, are prime candidates for testing speciation by host plant specialization. Here, we combined a phylogenetic and morphological approach with cross-pathogenicity tests to investigate host plant specialization and host range expansion in grapevine downy mildew. This destructive disease is caused by Plasmopara viticola, an oomycete endemic to North America on wild species and cultivated grapevines. Multiple genealogies and sporangia morphology provide evidence that P. viticola is a complex of four cryptic species, each associated with different host plants. Cross-inoculation experiments showed complete host plant specialization on Parthenocissus quinquefolia and on Vitis riparia, whereas cryptic species found on V. aestivalis, V. labrusca and V. vinifera were revealed to be less specific. We reconstructed the recent host range expansion of P. viticola from wild to cultivated grapevines, and showed that it was accompanied by an increase in aggressiveness of the pathogen. This case study on grapevine downy mildew illustrates how biotrophic plant pathogens can diversify by host plant specialization and emerge in agrosystems by shifting to cultivated hosts. These results might have important implications for viticulture, including breeding for resistance and disease management.