The Toll/interleukin-1 receptor nucleotide-binding site leucine-rich repeat gene, "resistance to Uncinula necator 1" (RUN1), from Vitis rotundifolia was recently identified and confirmed to confer resistance to the grapevine powdery mildew fungus Erysiphe necator (syn. U. necator) in transgenic V. vinifera cultivars. However, sporulating powdery mildew colonies and cleistothecia of the heterothallic pathogen have been found on introgression lines containing the RUN1 locus growing in New York (NY). Two E. necator isolates collected from RUN1 vines were designated NY1-131 and NY1-137 and were used in this study to inform a strategy for durable RUN1 deployment. In order to achieve this, fitness parameters of NY1-131 and NY1-137 were quantified relative to powdery mildew isolates collected from V. rotundifolia and V. vinifera on vines containing alleles of the powdery mildew resistance genes RUN1, RUN2, or REN2. The results clearly demonstrate the race specificity of RUN1, RUN2, and REN2 resistance alleles, all of which exhibit programmed cell death (PCD)-mediated resistance. The NY1 isolates investigated were found to have an intermediate virulence on RUN1 vines, although this may be allele specific, while the Musc4 isolate collected from V. rotundifolia was virulent on all RUN1 vines. Another powdery mildew resistance locus, RUN2, was previously mapped in different V. rotundifolia genotypes, and two alleles (RUN2.1 and RUN2.2) were identified. The RUN2.1 allele was found to provide PCD-mediated resistance to both an NY1 isolate and Musc4. Importantly, REN2 vines were resistant to the NY1 isolates and RUN1REN2 vines combining both genes displayed additional resistance. Based on these results, RUN1-mediated resistance in grapevine may be enhanced by pyramiding with RUN2.1 or REN2; however, naturally occurring isolates in North America display some virulence on vines with these resistance genes. The characterization of additional resistance sources is needed to identify resistance gene combinations that will further enhance durability. For the resistance gene combinations currently available, we recommend using complementary management strategies, including fungicide application, to reduce populations of virulent isolates.