The universal advantages of fermentation technology at high temperature of which thermotolerant ethanologenic yeasts play an essential role are cost reduction associated with cooling fermentation and lower risk of bacterial contamination. The aim of this study is to select the useful thermotolerant ethanologenic yeasts for bioethanol production at high temperature. In this study, ethanologenic yeasts isolated and purified from many different kinds of raw material sources in the Mekong Delta, Vietnam, including soil samples and other kinds of samples such as fruits, flowers, cocoa, fermented products, alcoholic rice starters, sugar-cane, molasses, and agricultural by-products. A total of 174 ethanologenic yeasts isolated from soil samples was examined for thermo-tolerance (37ºC, 40ºC, 43ºC, and 45ºC), and ethanol tolerance (8, 10, 12, and 14% (v/v) of ethanol). The preliminary test for ethanol production was performed in 10 mL of YM medium containing 16% (w/v) of glucose at 37ºC, and the followed up experiment was done in 250-mL flasks with 100 mL of the same medium at 37ºC, 40ºC, and 45ºC. The results showed that approximately 90.80% and 77.59% of yeast isolates could grow at 40ºC and 43ºC, respectively, but only 11.49% (20/174) of yeast isolates could grow at 45ºC. There were 35.63% and 17.24% of yeast isolates being able to grow in the medium containing 12% and 14% (v/v) of ethanol, respectively. Preliminary study on ethanol fermentation ability of 57 target isolates which grew at 40ºC as well as at 12% (v/v) of ethanol revealed that they could produce 23.15-55.69 g/L ethanol after 60 hours of fermentation at 37ºC. Ethanol concentration, volumetric ethanol productivity and fermentation efficiency of 8 selected isolates (BL5.1, CM4.2, CT5.3, DT5.3, HG1.1, HG1.2, ST1.1 and TG5.3) were as follow: 76.43-78.96 g/L, 1.06-1.30 g/L.h, and 93.99-96.76% at 37ºC; and 56.07-76.80 g/L, 1.01-1.60 g/L.h, and 68.71-94.12% at 40ºC, respectively. At 45ºC, maximum ethanol concentration and fermentation efficiency were not statistically significant difference between two predominant isolates (CM4.2 and ST1.1). In addition, a collection of 387 yeasts isolated from other kinds of samples were also examined for thermo-tolerant and ethanol-tolerant ability. The yeast isolates with high thermo- and ethanol tolerant ability were screened in 2% (w/v) glucose and fermented at 30ºC, 35ºC, 37ºC, 40ºC and 42ºC in molasses medium (22ºBrix). There were 84% of isolates could grow at 40ºC, and only 35.9% being able to grow in the medium supplemented with 12% (v/v) of ethanol. Saccharomyces cerevisiae was the most abundant identified species and had the highest thermo- and ethanol tolerant ability, and ethanol production in molasses medium. The ethanol concentrations were 7.21-8.05% (v/v) at 30oC, 7.02-8.18% (v/v) at 35oC, 5.67-8.12% (v/v) at 37oC, 2.77-6.26% (v/v) at 40oC, and 2.69-3.79% (v/v) at 42oC for 5 days of fermentation. Identification, biodiversity and ethanol production efficiency of these selected isolates are now under our investigation.