Abstract: In order to screen thermotolerant yeasts from special natural environment for ethanol fermentation， we carried out this study. After obtaining the isolates with traditional microbiological methods，we performed idenfication for the isolates according to morphological，physiological and biochemistrial characteristics，integrated 26S rDNA D1/D2 section sequences comparison and phylogenenitic analysis. Furthermore，we investigated the thermotolerance，acidfastness，sugar tolerance，ethanol tolerance and ethanol yield of the isolates by rutine methods. We obtained two thermotolerant yeasts from the soils for Moutai wine lees stacking，named Y1 and Y2. The Y1 and Y2 were classified to Issatchenkia orientalis and Pichia kudriavzevii，respectively. The tolerance test results showed that isolates Y1 and Y2 both could survive at 45℃，whereas they showed poor growth and abnormal colonies when exceeded 45℃. They showed growth inhibility when initial sugar concentration was up to 25%，and also the same when initial ethanol concentration was up to 8% and 10% for Y1 and Y2. The optimal pH for them was 4. The ethanol yields of Y1 and Y2 were 3.8% and 8.7% at 30℃，and 5.1% and 9.8% at 45℃，respectively. The ethanol tolerance and fermentation capabilty under the two temperature of 30℃ and 45℃ of Y2 was more excellent than Y1. So we deduced that the isolate yeast Y1 maybe used in simultaneous saccharification and fermentation.