The thermotolerant strain Saccharomyces cerevisiae DQ1 was applied to the simultaneous saccharification and fermentation (SSF) at high temperature and high solids loading of the dilute acid-pretreated corn stover in the present study. The SSF using S. cerevisiae DQ1 was operated at 30 % solids loading of the pretreated corn stover with three-step SSF mode and achieved up to ethanol titer of 48 g/L and yield of 65.6 %. S. cerevisiae DQ1 showed strong thermotolerance in both the regular one-step SSF and the three-step SSF with changing temperature in each step. The three-step SSF at 40°C using S. cerevisiae DQ1 tolerated the greater cellulase dosage and solids loading of the pretreated corn stover and resulted in increased ethanol production. The present study provided a practical potential for the future SSF of lignocellulose feedstock at high temperature to reach high ethanol titer.