Although some engineered S. cerevisiae strains exhibit good xylose utilization ability, the lack of tolerance to inhibitors generated in biomass pretreatment limits the application of such strains in the production of bioethanol from lignocellulosic biomass. By applying a sexual mating method, inhibitor tolerance was developed in xylose-utilizing strains. The final ethanol concentrations in simultaneous scarification and co-fermentation (SScF) process at 38 °C with hybrid strains were 50% higher than the SScF process with the xylose-fermenting parent strain. The strain viability of the hybrid strain E7-12 at 24 h was 282 times higher than the parent strain in the SScF process at 25% solid loading. Due to the improved sugar utilization, the final ethanol concentration reached 69.7 g/L (E7-11) and 70.0 g/L (E7-12), which were 25.3 g/L and 25.6 g/L higher than that of SScF with the xylose-fermenting strain, respectively.