The grape powdery mildew pathogen (Erysiphe necator), has a center of diversity in eastern North America. Genetic divergence of E. necator populations on native or introduced Vitis species in this region has not been reported in previous studies, except on muscadine grapes (V. rotundifolia). In this study, we used AmpSeq to genotype 6 polymorphic microsatellite markers to quantify the genetic diversity of E. necator populations in 3 commercial V. vinifera vineyards, a commercial vineyard of the interspecific hybrid ‘Chancellor’, and a vineyard hosting fungicide efficacy trials on V. vinifera ‘Chardonnay.’ Across 300 samples, AmpSeq returned an average of 7.7 alleles per marker compared to 6.2 for the same markers assayed by capillary electrophoresis in a previous study of 129 samples spanning the Eastern United States and Europe. Multidimensional scaling plots along with pairwise F_ST values ranging from 0.00 to 0.02 suggested that isolates collected from various fungicide treatments in the efficacy trial vineyard had the lowest within-site diversity. Across all markers, no private alleles were identified in isolates from fungicide trial treatments, compared to 3 across V. vinifera commercial sites and 12 in the Chancellor site. These preliminary data suggest that isolates from the interspecific hybrid vineyard were the most genetically diverged. Further studies are needed to determine how geography, fungicide use, and host impact the pathogen population.