Nowadays, vinegar is industrially produced by mesophilic acetic acid bacteria (AAB). However, temperature fluctuation during acetous fermentation is inevitable, and may cause process disturbances. This can be mostly avoided using thermo-tolerant AAB. The main purpose of the present study was to isolate thermo-tolerant AAB. Fermentation performances were then evaluated. Twenty-eight different isolates were isolated from Iranian traditional vinegar. One of the isolates was able to grow and produce acetic acid in minimal culture media containing 5% (w/v) ethanol at 30-42 ºC. 16s rRNA gene analysis showed that the selected thermo-tolerant isolate was Acetobacter tropicalis (L31). Acetous fermentation in Lab-bioreactor showed that Acetobacter tropicalis (L31) grew in minimal culture medium, and produced 5% (w/v) acetic acid at 37°C in batch and semi-continuous fermentation mode. Fermentation time was significantly dependent on dissolved oxygen (DO) concentration and acclimation of cells to low pH and acid stress. In average, acclimated cells produced 2.3-2.7 g L-1h-1 acetic acid during production phase. The final yield was 87% at 37ºC in low and high DO concentrations. Low DO concentration (15%) during acetous fermentation caused longer fermentation time, but a large part of cells (91%) grown under such condition was viable if oxygen flow was interrupted. In contrast, cells grown under high DO concentration were not able to tolerate oxygen deficiency. In conclusion, since Acetobacter tropicalis (L31) grew well in minimal culture medium, and showed tolerance to high temperature and low DO, it seems that it is a potential isolate for vinegar starter production.