Erysiphe necator populations, causing powdery mildew of grapes, have a complex genetic structure. Two genotypes, A and B, were identified in most vineyards across the world on the basis of fixed single nucleotide polymorphisms (SNPs) in several DNA regions. It was hypothesized that A populations overwinter as mycelia in grapevine buds, giving rise to so-called flag shoots in spring, and are more sensitive to fungicides than B populations, which overwinter as ascospores and become widespread later in the season. Other studies concluded that the biological significance of these genotypes is unclear. In the spring of 2015, there was a unique opportunity to collect E. necator samples from flag shoots in Hungary. The same grapevines were sampled in summer and autumn as well. A total of 182 samples were genotyped on the basis of β-tubulin (TUB2), nuclear ribosomal DNA (nrDNA) intergenic spacer (IGS), and internal transcribed spacer (ITS) sequences. Genotypes of 56 samples collected in 2009–2011 were used for comparison. Genotype A was not detected at all in spring, and was present in only 19 samples in total, mixed with genotype B, and sometimes with another frequently found genotype, designated as B2. These results did not support the hypothesis about temporal isolation of the two genotypes and indicated that these are randomly distributed in vineyards.