Hop powdery mildew (Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. Post 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in cv. ‘Cascade’. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race or races may be present on susceptible cultivars and other germplasm. Current methods for race determination for P. macularis are slow, costly, and labor intensive. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, dubbed V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6-isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short indels. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting multiplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with other powdery mildew species and pathogens of hop. Weak non-specific amplification occurred with powdery mildew collected from grape, strawberry, and zinnia; however, non-specification amplification is not a concern when differentiating pathogen race from mildew colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.