The development of hybrids has been an effective approach to generate novel yeast strains with optimal technological profile for use in beer production. This study describes the generation of a new yeast strain for lager beer production by direct mating between two Saccharomyces cerevisiae strains isolated from cachaça distilleries: one that was strongly flocculent, and the other with higher production of acetate esters. The first step in this procedure was to analyze the sporulation ability and reproductive cycle of strains belonging to a specific collection of yeasts isolated from cachaça fermen- tation vats. Most strains showed high rates of sporulation, spore viability, and homothal- lic behavior. In order to obtain new yeast strains with desirable properties useful for la- ger beer production, we compare haploid-to-haploid and diploid-to-diploid mating procedures. Moreover, an assessment of parental phenotype traits showed that the seg- regant diploid C2-1d generated from a diploid-to-diploid mating experiment showed good fermentation performance at low temperature, high flocculation capacity, and de- sirable production of acetate esters that was significantly better than that of one type la- ger strain. Therefore, strain C2-1d might be an important candidate for the production of lager beer, with distinct fruit traces and originating using a non-genetically modified organism (GMO) approach. Recent work has suggested the utilization of hybridization techniques for the generation of novel non-genetically modified brewing yeast strains with combined properties not commonly found in a unique yeast strain. We have observed remarkable traits, especially low temperature tolerance, maltotriose utilization, flocculation ability, and production of volatile aroma compounds, among a collection of Saccharomyces cerevisiae strains isolated from cachaça distilleries, which allow their utilization in the production of beer. The significance of our research is in the use of breeding/hybridization techniques to generate yeast strains that would be appropriate for producing new lager beers by exploring the capacity of cachaça yeast strains to flocculate and to ferment maltose at low temperature, with the concomitant production of flavoring compounds.