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Obligately biotrophic plant pathogens pose challenges in population genetic studies due to their genomic complexities and elaborate culturing requirements with limited biomass. Hop powdery mildew (Podosphaera macula, is) is an obligately biotrophic ascomycete that threatens sustainable hop production. P macularis populations of the Pacific Northwest (PNW) United States differ from those of the Midwest and Northeastern United States, lacking one of two mating types needed for sexual recombination highand harboring two strains that are differentially aggressive on the cultivar Cascade and able to overcome the Humulus lupulus R-gene R6 (V6), respectively. To develop a high-throughput marker platform for tracking the flow of genotypes across the United States and internationally, we used an existing transcriptome of diverse P macularis isolates to design a multiplex 54 amplicon sequencing markers, validated across a panel of 391 U.S. samples and 123 international samples. The results suggest that niacu[£iris from U.S. commercial hop yards form one population closely related to P macularis of the United Kingdom, while P m Licul(iris from U.S. feral hop locations grouped with P macul(iris of Eastern Europe. Included in this multiplex was a marker that successfully tracked V6-vii·ulence in 65 of 66 samples with a confirmed V6-phenotype. A new qPCR assay for high-throughput genotyping of P macuknis mating type generated the highest resolution distribution map of' P mcicularis mating type to date. Together, these genotyping strategies enable the highand throughput and inexpensive tracking of pathogen spread among geographical regions from single-colony samples and provide a roadmap to develop markers for other obligate biotrophs. [ABSTRACT FROM AUTHOR]