Explorational research of useful thermotolerant ethanologenic yeasts and their applications for the development of next-generation fermentation technology have been increasingly considered world-wide. In this study, a collection of 387 ethanologenic yeasts isolated and purified from many different kinds of raw material sources in the Mekong Delta (including about 9 cities and provinces) of Vietnam; such as fruits, flowers, cocoa, fermented products, alcoholic rice starters, sugar-cane, molasses, soil samples and agricultural by-products. The microbial population and the characteristics of morphology, physiology, biochemistry of these yeasts were examined and recorded. All isolates were examined for growth at 30, 35, 37, 40, 43, and 45ºC, and for ethanol tolerant ability at 3, 6, 9, and 12% v/v of ethanol. The yeast isolates with high thermo- and ethanol tolerant ability were screened in 2% w/v glucose and fermented at 30, 35, 37, 40 and 42ºC in molasses medium (22ºBrix). T5 Based on characteristics of morphology, physiology and biochemistry, yeast isolates were classified into some genera including Saccharomyces, Kluyveromyces, Candida, Pichia, Torulaspora, and Hanseniaspora. Eighty four percent of yeast isolates could grow at 40ºC but only 56.4% and 8.6% of yeast isolates could grow at 43ºC and 45ºC, respectively. More than 80% of yeast isolates were able to grow in the medium containing 9% v/v of ethanol and it was decreased to 35.9% of yeast isolates in the medium supplemented 12% v/v of ethanol. The genetic characterization of the target selected strains has been preliminarily implemented. Results of the molecular analysis of ITS1, 5.8S rDNA, and ITS2 sequence showed that Saccharomyces cerevisiae was the most abundant species. S. cerevisiae strains isolated from alcoholic rice starters were found to perform the highest thermo- and ethanol tolerant ability, and ethanol production in molasses medium. The ethanol concentrations were 7.21-8.05% at 30^oC, 7.02-8.18% (v/v) at 35^oC, 5.67-8.12% (v/v) at 37^oC, 2.77-6.26% (v/v) at 40^oC, and 2.69-3.79% (v/v) at 42^oC for 5 days of fermentation. The genetic biodiversity of selected predominant isolates are now in progress, and their application for ethanol fermentation at high temperature at the laboratory-scale and the pilot-scale testing trial will be further followed up.