Aims: To isolate thermotolerant Saccharomyces cerevisiae with high-energy-pulse-electron (HEPE) beam, to optimize the mutation strain fermentation conditions for ethanol production and to conduct a preliminary investigation into the thermotolerant mechanisms. Methods and Results: After HEPE beam radiation, the thermotolerant S. cerevisiae strain Y43 was obtained at 45 degrees C. Moreover, the fermentation conditions of mutant Y43 were optimized by L3 3 orthogonal experiment. The optimal glucose content and initial pH for fermentation were 20% g(-1) and 4 5, respectively; peptone content was the most neglected important factor. Under this condition, ethanol production of Y43 was 83 1 g(-1) after fermentation for 48 h at 43 degrees C, and ethanol yield was 0 42 g g(-1), which was about 81 5% of the theoretical yield. The results also showed that the trehalose content and the expression of the genes MSN2, SSA3 and TPS1 in Y43 were higher than those in the original strain (YE0) under the same stress conditions. Conclusions: A genetically stable mutant strain with high ethanol yield under heat stress was obtained using HEPE. This mutant may be a suitable candidate for the industrial-scale ethanol production. Significance and Impact of the Study: High-energy-pulse-electron radiation is a new efficient technology in breeding micro-organisms. The mutant obtained in this work has the advantages in industrial ethanol production under thermostress.