Lignocellulosic biomass is a highly promising source for the generation of sustainable energy. Bioethanol produced from lignocellulosic biomass can be used to fuel internal combustion engines that run on gasoline. To reduce the cost of ethanol production from lignocellulosic biomass, the simultaneous saccharification and fermentation (SSF) method is attractive, because it can be performed in a single reactor. We attempted to isolate thermo tolerant and fermentation inhibitor-tolerant strains of yeast by altering the preculture conditions. Preculture at 35 increased SSF ethanol production in the presence of 5-HMF. This study demonstrated that yeast strains can adapt to higher preculture temperature for SSF.