Aims: To study the diversity and dynamics of indigenous Saccharomyces wine populations during Malbec spontaneous fermentation, a representative Patagonian red wine, at both industrial and laboratory scale. Methods and Results: Two molecular techniques, including restriction fragment length polymorphism of mitochondrial (mt) DNA and polymorphism of amplified delta interspersed element sequences, were used for characterization of indigenous yeasts at strain level. The mtDNA restriction patterns showed the major discriminative power; however, by combining the two molecular approaches it was possible to distinguish a larger number of strains and, therefore, draw more representative conclusions about yeast diversity. Although a great diversity of wild Saccharomyces cerevisiae strains was observed, only nine represented more than half of the total Saccharomyces yeast biota analysed; five of these were common and took over the Malbec must fermentation in both vinifications. Conclusions: Many different indigenous S. cerevisiae strains were identified; nevertheless, the dominant strains in both industrial and laboratory vinification processes were just a few and the same. Significance and Impact of the Study: Small-scale fermentation appears to be a valuable tool in winemaking, one especially helpful in evaluating microbiological aspects of as well as possible interactions between inoculated selected strains and native strains.