Saccharomyces cerevisiae strains isolated from wine, rum, bread, or oak present a wide phenotypic divergence (1, 3). Different genome sequencing programs by our group (2,3) and others (4) have offered clues about the different mechanisms permitting the adaptation of yeast to wine environment. Copy number variations, translocations, and horizontal transfers have been shown to participate in the adaptation of Saccharomyces cerevisiae to the grape must (3,5). In contrast to the flood of genomic data, we have very few experimental clues on how genetic variation participated in the evolution of wine yeast, and experimental evolution is the method of choice to investigate such questions. To understand how yeast adapted to the wine environment, we built 3 x 4 recombinant populations containing either wine strains, Mediterranean oak strains (as a grape must naive population), and strains from both origins. These three populations were grown, them for 24 fermentations in a Sauvignon grape must that contained assimilable nitrogen and sterol at a non-limiting concentrations. In addition, the potential effect of the grape must microbiota was evaluated through the addition of a synthetic microflore. After the evolution, evolved strains were phenotyped for their ability to grow in the grape must and ferment and compared to the ancestral population. As expected, all populations performed better at the end of the experiment, and populations containing wine alleles outperformed the Mediterranean oak populations. The effect of the presence of a synthetic microflore could only be slightly observed on the maximum fermentation speed. The comparison of the genome of adapted populations to the ancestral pool, revealed shifts in allelic frequencies in regions associated to specific molecular functions or cellular compartment in the three adapted lineages. However, mitochondrial genes were impacted in all adapted lineages. Interestingly, when considering the Mediterranean oak/ wine populations, the genes SSU1 and ECM34 involved in sulfite resistance (5) or MDS3 and GCN1 involved in nitrogen management (6) were found to participate in the adaptation to the grape. This highlights the complexity of the adaptation to the grape must.