High temperature and high ethanol concentrations obviously affect vinegar fermentation. The thermotolerant and ethanol-resistant strains are expected to become one of the technologies for effective vinegar fermentation. This study aimed to further improve thermotolerant Komagataeibacter oboediens MSKU 3 through thermal and ethanol adaptations for acetic acid fermentation. The MSKU 3 strain was independently cultured by a repetitive cultivation in gradually increasing temperature from 37 to 39 degrees C for thermal adaptation, while adaptation to ethanol was carried out from concentrations of 3 to 5.5% (v/v) at 37 degrees C. Acetic acid fermentation revealed that the thermo-adapted T4 strain could produce 2.82% acidity with 3% ethanol at 39 degrees C, whereas the ethanol-adapted E3 strain could produce 3.54% acidity with 5.5% ethanol at 37 degrees C, in contrast to the parental strain, MSKU 3, in which no fermentation occurs at either 39 degrees C or 5.5% ethanol. Furthermore, genome mapping analysis of T4 and E3 strains against the genome of parental strain MSKU 3 revealed several mutated genes that are associated with thermotolerance or ethanol adaptation. The occurrence of these adaptation-associated mutations during adaptive evolution was also analyzed. Therefore, adapted strains T4 and E3 revealed the potential of Komagataeibacter oboediens strain improvement to further enhance vinegar fermentation with high ethanol concentration at high temperature.