Isolated yeast populations of Chardonnay grape must during spontaneous fermentation were compared to those isolated on grape berries and in a winery environment before the arrival of the harvest (air, floor, winery equipment) and in the air through time. Two genera of yeast, Hanseniaspora and Saccharomyces, were isolated in grape must and in the winery environment before the arrival of the harvest but not on grape berries. The genus Hanseniaspora represented 27% of isolates in the must and 35% of isolates in the winery environment. The isolates of these two species were discriminated at the strain level by Fourier transform infrared spectroscopy. The diversity of these strains observed in the winery environment (26 strains) and in must (12 strains) was considerable. 58% of the yeasts of the genus Hanseniaspora isolated in the must corresponded to strains present in the winery before the arrival of the harvest. Although the proportion and number of strains of the genus Hanseniaspora decreased during fermentation, some strains, all from the winery environment, subsisted up to 5% ethanol content. This is the first time that the implantation in grape must of populations present in the winery environment has been demonstrated for a non-Saccharomyces genus.