Aims: To test indigenous Saccharomyces cerevisiae as starters to produce cachaca in large-scale in a traditional distillery, establishing the period in which, each strain predominates in the vats, chemical composition and sensory attributes of the beverage, and to compare these data with vats prepared by spontaneous fermentation. Methods and Results: Strains were evaluated for kinetic fermentation parameters, permanence in vats, volatile compound production, and sensory attributes for the cachacas produced. In general the vats in which starter strains were used, no difference in restriction mitochondrial DNA (mtDNA) profiles of isolates was observed. In the vats in which spontaneous fermentation occurred, different mtDNA restriction profiles were observed. Most of the non-Saccharomyces species isolated could be regarded as contaminants of fermentation. All cachacas produced, despite being recently distilled and with differences in their chemical composition, were well accepted by the judges. Conclusions: It was possible to detect the differences in the fermentation capacities of S. cerevisiae strains, in their relative abundances at different time periods, and in the chemical compositions and sensory attributes of the resulting beverages. Significance and Impact of the Study: The indigenous strains utilized to prepare cachaca have shown potential to be used as starters of this traditional fermentation process.