Laser radiation is an efficient approach for rapid improvement of industrial microbial phenotypes. To improve ethanol tolerance in Saccharomyces cerevisiae strains, a 266 nm laser radiation with the use of repetitive cultivation was explored in this work. After irradiated by 266 nm laser radiation and repetitive cultivation, a genetically stable SM4 strain was obtained. The SM4 strain could grow on YPD plate with extra 15% (v/v) ethanol. Moreover, the ethanol production performance of SM4 strain was 29.25% more than that of the wild type strain when they were cultivated in 5% (v/v) ethanol fermentation medium for 72 h. The DNA mutation was the possible characters for the phenotype of SM4 strain. Overall, the 266 nm laser radiation and repetitive cultivation approach might be a novel and useful for breeding fermentation microorganisms. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.