For industrial applications, fermentation of ethanol at high temperature offers advantages such as reduction in cooling costs, reduced risk of microbial contamination and higher efficiency of fermentation processes including saccharification and continuous ethanol stripping. Three thermotolerant Saccharomyces cerevisiae isolates (C3723, C3751 and C3867) from Thai fruits were capable of growing and producing 38 g/L ethanol up to 41 degrees C. Based on genetic analyses, these isolates were prototrophic and homothallic, with dominant homothallic and thermotolerant phenotypes. After short-term (30 min) and long-term (12 h) exposure at 37 degrees C, expression levels increased for the heat stress-response genes HSP26, SSA4, HSP82, and HSP104 encoding the heat shock proteins small HSP, HSP70, HSP90 and the HSP100 family, respectively. In isolates 0723 and 0867, expression was significantly higher than that in reference isolates W303 and TISTR5606 for TPS1 encoding trehalose-6-phosphate synthase, NTH1 encoding neutral trehalase and GSY1 encoding glycogen synthase. The results suggested that continuous high expression of heat stress-response genes was important for the long-term, heat stress tolerance of these thermotolerant isolates. (c) 2012, The Society for Biotechnology, Japan. All rights reserved.