The study on exploration of useful thermotolerant microorganisms and their applications for fermentation technology at high temperature have been increasingly considered world-wide. In this research project, a collection of 762 isolates (including 387 yeasts, 178 acetic acid bacteria, and 197 lactic acid bacteria) was purified from many different kinds of raw material sources in the Mekong Delta, Vietnam; such as fruits, flowers, cocoa, fermented products, alcoholic rice starters, sugarcane, molasses, agricultural by-products and soil samples. For yeasts, there were 84% of yeasts could grow at 40ºC but only 56.4% and 8.6% of yeasts could grow at 43ºC and 45ºC, respectively. More than 80% of yeasts were able to grow in the medium containing 9% (v/v) of ethanol and it was decreased to 35.9% of yeasts growing in the medium supplemented 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% 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. For acetic acid bacteria, the results of isolation and characterization showed that 97.2% of isolates could grow at 39ºC. The percentage of growing decreased to 88.0%, 66.2% and 37.3% when the incubation temperatures increased to 41, 43 and 45ºC, respectively. A half of tested isolates could grow in the media supplemented with 1.0% (v/v) of acetic acid and it was decreased to 20.4%, 9.86%, 2.82% and 2.11% of isolates in the medium containing 1.5%, 2.0%, 2.5% and 3.0% (v/v) of acetic acid, respectively. The isolates from fermenting cocoa pulp had the highest fermented capacity and be stable with the increasing of temperature. The acetic acid concentrations could reach to 3.32-6.48%, 4.08-6.28%, 3.24-6.68% and 3.84-6.53% (w/v) at 30, 37, 38 and 39ºC, respectively. The identification result of the 3 superior isolates was Acetobacter pasteurianus. For lactic acid bacteria, the results revealved that almost isolates could grow at 41oC (92,57%). The percentage of growing decreased to 79.73%, 61.14%, and 18.92% when the incubation temperatures increased to 43oC, 45oC, and 47oC, respectively. The average diameters of clear zone for acid producing capacity and antimicrobial activity tests on agar media were 11.71 mm and 7.83 mm, respectively. The results of acid production were recorded as follow: 0.60-1.50% (w/v) at 37oC, 0.75-1.73% (w/v) at 38oC, 0.68-1.35% (w/v) at 39oC, and 0.30-1.20% (w/v) at 40oC. The genetic diversity and application of selected useful thermotolerant isolates for fermentation of ethanol, acetic acid and lactic acid at the laboratory-scale and the pilot-scale testing trials are now in progress.