Powdery mildew, caused by the fungus Erysiphe necator, is a constant threat to worldwide grape production. Management requires the repeated use of target-specific fungicides. Resistance to commonly used quinone outside inhibitors (QoIs) and demethylation inhibitors (DMIs) fungicides is known to occur in subpopulations of E. necator and is associated with single nucleotide polymorphisms (SNPs) in the cytochrome b and cyp51 genes, respectively. DMI resistance in E. necator is further complicated by cyp51 copy number variation. To track this resistance, two TaqMan-based monitoring tools were developed for both QoI and DMI resistance loci. For cyp51, a marker was developed to monitor both SNP and copy number variation simultaneously. Specificity and sensitivity of these assays showed that these tools could discriminate SNPs and were effective on mixed samples. We implemented these diagnostic tools to survey E. necator isolates collected from leaf and air samples from major grape growing areas in Washington, Oregon and California and findings were corroborated with EC50 values from fungicide-amended agar germination bioassays in the case of the cytochrome b gene and QoI resistance. Our findings showed that results from these TaqMan probe-based allele-specific assays and bioassays are highly correlated. Overall, these diagnostic tools will help grape growers make informed decisions on fungicide selections and applications, which in turn, will facilitate grapevine powdery mildew disease management and improved grape production systems.