Aims The yeast community structure associated with grapes is an essential part of the wine‐growing chain with a significant effect on wine quality. The aim of the present study was to evaluate the effect of the varietal factor on the yeast community assembly on grapes and during must fermentation. Methods and Results We analysed the wine yeast populations associated with four different grape varieties from the Greek national collection vineyard of Lykovryssi. The vintage effect was also considered by sampling the grapes for two consecutive years. Fourteen yeast species were recovered and genotyped to distinct subpopulations. A relatively stable yeast community structure was detected across vintages, with Hanseniaspora guilliermondii being the core species of the vineyard under study. The detected species subpopulations shared a relatively high genetic similarity with several genotypes persisting across vintages. Conclusions It was shown that different grape cultivars were associated with distinct yeast communities, pointing to their possible implication on wine chemical diversity. Significance and Impact of the Study Present findings show that the varietal factor is an important sharpener of the vineyard‐associated wine yeast community, which may interfere with the organoleptic profile of the resulting wines.