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Winemakers are increasingly keen to limit the use of commercial yeasts in order to reduce oenological inputs. The preparation of an indigenous winery-made fermentation starter from grapes called ‘pied de cuve’ (PdC) is becoming popular, especially in organic farming systems. However, the implementation of the PdC method is still empirical and knowledge is lacking regarding the impact of PdC on S. cerevisiae diversity during alcoholic fermentation. In this study, the impact of PdC on S. cerevisiae genetic diversity and wine composition was evaluated at an industrial scale. Despite very low initial population level of S. cerevisiae before inoculation, the use of PdC was as efficient as Active Dry Yeast in terms of fermentation kinetics and chemical analyses on the resulting wines, except for one modality. At mid-fermentation, the diversity of S. cerevisiae strains was different depending on the PdC used, and was also different from that in the spontaneous fermentation with, in some cases, clonal expansion. Our results provide evidence that the use of PdC could secure the fermentation process more efficiently than spontaneous fermentation.