Vinegar is a valuable food additive and preservative effective against food spoilage. Acetobacters are the most important bacteria in vinegar with significant worldwide importance in the vinegar production industry. Therefore, many studies have been done on these bacteria. This study aimed to isolate and identify acetic acid bacteria from vinegar by common bacterial culture methods and sequencing of the 16S rRNA gene. To isolate Acetobacter from vinegar, 13 samples of different types of vinegar (made from grape, apple, and berry) were collected from various parts of Isfahan and transferred to the laboratory. The samples were cultured on specific media and identified using macroscopic and microscopic observations, and PCR through 16S rRNA gene sequencing. Among the investigated samples, 3 Acetobacter samples were isolated. After performing PCR and comparing the sequence of the PCR product of all three 16S rDNA genes of bacteria with the data available in the NCBI gene bank, 99% similarity with Acetobacter pasteurianus was shown. By isolating Acetobacter from different types of vinegar, it is possible to investigate their ability to grow at high temperatures and with varying amounts of acetic acid and ethanol at various temperatures for the industrial applications of these bacteria.