Nowadays, thermotolerant ethanologenic yeasts have attracted many scientists due to the current challenges of increasing global temperature. There are several advantages associated with using thermotolerant yeasts in many different aspects of fermentation technology, especially processing at high temperature, reducing cooling cost, and lessening risk of contamination. The aims of this study are to isolate, characterize and select useful thermotolerant ethanologenic yeasts for bioethanol production at high temperature. In this study, a collection of 174 ethanologenic yeasts was isolated from 80 soil samples in 13 provinces of Mekong Delta, Vietnam, by using enrichment culture technique carried out at 35ºC. Yeast isolates were tested for morphological and biochemical characteristics. All isolates were examined for growth at 37, 40, 43, and 45ºC and for ethanol tolerance ability at 8, 10, 12, and 14% (v/v) of ethanol, and screened in 2% (w/v) D-xylose at 35ºC. The results showed that most of the yeast’s cells are spherical, oval and ellipse, single or linked in association, size range (1.5-3.0) µm x (3.0-7.0) µm. Almost of yeast isolates could grow well at elevated temperature up to 40ºC. Approximately 90.8% and 77.6% of yeast isolates could grow at 40ºC and 43ºC, respectively, but only 11.5% of yeast isolates could grow at 45ºC. About 86.8% of yeast isolates were able to grow in the medium containing 10% (v/v) of ethanol; however, in the media supplemented with 12% and 14% (v/v) of ethanol, the growth rate decreased to 35.6% and 17.2%, respectively. Preliminary study on xylose fermentation ability revealed that all newly yeast isolates were unable to ferment xylose, however more than 70% of them could consume this sugar. By screening of the thermo- and ethanol tolerance ability as well as the xylose-utilizing capacity, 27 yeast isolates had been selected for their superior thermo-tolerance (up to 40°C) and ethanol tolerance (up to 14% (v/v) ethanol). These selected isolates have been identifying by molecular techniques and characterizing the fermentation capacity at different temperatures for further experiments.