Queretaro is the second state in wine production in Mexico, but the commercialization is difficult because of the lack of quality and tipicity. An alternative is to have native yeasts selected to increase the potential of grape juices and provide originality to the wines. The objective of this study was the isolation, selection based on their wine potential, and identification of native Saccharomyces yeasts in Queretaro vineyards. The strains were isolated in 2012 at the physiology and fermentations laboratory, Universidad Autemoma de Queretaro, Mexico, during the spontaneous fermentation of juices from mature grapes collected randomly inside three Queretaro vineyards, differentiating the Saccharomyces in a lysine medium. Their tolerance to ethanol (12 %), sulfite addition (200 mg L-1) and the killer phenotype was evaluated using Bioscreen; their enological potential was determined in microvinification tests with Cabernet Sauvignon and Merlot varieties, and they were identified by amplifying the D1/D2 domain. Eight strains stood out because of their tolerance to SO2, ethanol and the killer phenotype: S. cereviasiae (SR19, SR26, 5R27, N05) and S. paradoxus (N42, SR25, OBIO, OB11). The wines produced with these yeasts were dry (<3.8 g glucose L-1), with total acidity of 6.5 to 8.4 g L-1) tartaric acid, volatile acidity of 0.13 to 0.26 g L-1 acetic acid, and pH of 3.56 to 3.74, without differences between yeasts (Tukey, p > 0.05). The native N05 and K1 (control) strains obtained the highest alcohol degrees (15.2 and 14.9 %), conversion efficiency (16.31 and 16.67 g glucose degrees alcohol(-1)) and fermentative efficiency (90.8 and 89.1 %). The SR26 strain contrasted with OB10 in the fermentation speed (19.2 vs. 18.6 g CO2 48 h1), and SR19 and SR26 obtained the lowest SO2 production (25.6 and 36.8 mg L1). This study revealed the potential of native yeasts from the state of Queretaro to be used in the production of regional wines.