Numerous ecological studies have been conducted over the years to understand the dynamics, quantification, and composition of the microflora responsible for spontaneous fermentation. Among them, yeasts are microorganisms that exert key processes and are responsible for alcoholic fermentation. In the present study, we isolated and characterized molecularly two yeasts by RFLP (Restriction Fragment Length Polymorphisms) in which we compared the restriction patterns of different genomic regions corresponding to the ribosomal DNA. Experimental design (ED) was based on the Response Surface Methodology (MSR) used to design a suitable culture medium for the production of Mezcal using as substrate an Agave cupreata extract juice and a LEVM yeast strain. We found by RFLP two Saccharomyces cerevisiae yeasts strains (LEVM y LEVZ). In ED for the LEVM the variables selected such as the pH, initial substrate concentration, and temperature were operating levels as close as possible to the original process, these preliminary results show the importance of using molecular techniques for the characterization of yeast strains used in the beverage industry and the use of ED allowed establish the fermentation process conditions.