During the spontaneous alcoholic fermentation there is a succession of different strains over the course of winemaking. The aim of the present study was to select a yeast strain with appropriate enological qualities and adapted to the ecological environment of the cellar to be used as starter culture. For that purpose yeast strains were isolated at different stages of fermentation. A total of 240 Saccharomyces spp isolates were characterized by DNA mitochondrial restriction analysis, which delivered 21 different molecular profiles which were then evaluated.Enological characteristics, nitrogen requirements, protein haze stabilization, and enzymatic activities were evaluated. Microvinifications were carried out with the finalist strains and chemical and sensory analyses were performed for selecting the most adequate Saccharomyces strain for that winery. Finally, two selected strains were used by the wine cellar in two consecutives vintages.