Currently, the state of Queretaro is a major wine producer in Mexico; however, marketing these wines is difficult due to their lack of quality and typicity, factors in which wine yeasts play an essential role. Saccharomyes are responsible for ethanol production and non-Saccharomyes (nS) can provide metabolites that enhance wine quality, so their use in mixed cultures is of great interest. In this study, yeasts isolated from the spontaneous fermentation of grape musts from three local vineyards were differentiated as nS in lysine medium. Their β-glucosidase activity and SO2 (30 mg·L-1) and ethanol (6 %) tolerances were evaluated. Outstanding yeasts were tested in mixed microvinifications along with S. cerevisiae (K1-V1116) and identified through analysis of the 26S gene. Of 197 isolated strains, 146 were differentiated as nS; 90 showed βglucosidase activity and eight (NB1, NA4, NB27, NB31, NB39, NR77, NR90, NB108) were selected based on tolerance tests, being identified as belonging to the genus Hanseniaspora. The nS did not interfere with the fermentative activity of K1-V1116, finding no differences in the parameters evaluated between the wines obtained by mixed cultures and the control (K1-V1116). The values obtained for pH, total acidity, volatile acidity and alcohol were considered to be of acceptable quality for dry wines. The strain NB39 stood out for its glycerol production, obtaining the highest value (7.45 g·L-1). We conclude that there are nS yeast strains in the region with enological potential to be used at a commercial level.