Vinegar fermentation is essentially a two-step process comprising anaerobic conversion of sugars to ethanol and aerobic oxidation of ethanol to acetic acid. Vinegar can be produced from many juice extracts using yeast and acetic acid bacteria (AAB). This research identified the genetic relation between some thermotolerant AAB strains, and the factors that affected vinegar production from Acerola (Malpighia emarginata) juice at high temperatures. The interaction between yeast Saccharomyces cerevisiae and AAB was investigated. Yeast was fermented for one day before inoculating the AAB and fermenting the mash. Acid production was 2.67% (w/v). Eight thermotolerant AAB strains (A1, A3, A18, A19, A28, A31, A50, and A55) were selected which produced 3.68-4.24% (w/v) of total acid at 37°C. Strain A28 with acid levels of 2.84% (w/v) and 2.88% (w/v) was selected for fermentation at 38°C and 39°C, respectively. The 16S rDNA genes of the eight AAB strains were sequenced and compared with reference strains in GenBank. Results indicated that most of the strains were closely related to species in the genus Acetobacter including Acetobacter tropicalis, A. pasteurianus, A. sicerae and A. senegalensis. The highest acidity of Acerola vinegar fermentation was obtained for A. senegalensis A28 at 39°C, with 5.34% (w/v) and sugar concentration of 20.26°Brix, cell density of 10⁶ cells/mL, and pH 6.5.