Transcriptome sequences of the grape powdery mildew fungus Erysiphe necator were used to develop microsatellite markers (EST-SSRs) to study its relatively unexplored population structure in its centre of diversity in eastern North America. Screening the transcriptome sequences revealed 116 contigs with candidate microsatellites, from which 11 polymorphic microsatellite markers were developed from 31 markers tested. Eight of these markers were used to genotype isolates from different regions and hosts in the eastern USA and compare them to samples from southern France and Italy. Genetic diversity in the eastern USA is much greater than in Europe. Bayesian cluster analyses showed that 10 isolates from North America have high affinities with, but differ from, European group A; these are referred to as A-like isolates. No isolates with close affinity to European group B were found in the eastern USA. Bayesian analyses also detected genetic differentiation between isolates from Vitis rotundifolia and isolates from other Vitis hosts. Genetic differentiation detected between the northeastern and southeastern USA was mostly attributable to the A-like isolates in the southeast, which are significantly more aggressive than the other populations. This research demonstrates that transcriptome sequencing of fungal pathogens is useful for developing genetic markers in protein-coding regions and highlights the role of these markers in population biology studies of E. necator.