Here, thermotolerant yeast mutants were screened through random respiration-deficient mutagenesis using ethidium bromide, which is known as mitochondrial mutagen in yeast. Next, Saccharomyces cerevisiae mb 2 among screened thermotolerant strains was selected as a suitable strain for bioethanol production at high temperature based on the growth and fermentation test in complex medium. Then, SSF was conducted to verify the feasibility of high temperature fermentation by Saccharomyces cerevisiae mb 2 using alkaline-pretreated Miscanthus containing 3-9 wt% glucan.