Identification and classification of strains of Acetobacter bacteria were determined by standard methods of microbiology and molecular biology. 16S-23S rDNA internal transcribed spacer regions of four strains of genus Acetobacter pasteurianus were analysed in order to re-examine classification of these strains. By ITS-PCR amplification of the 16S?23S spacers, two products of similar size were produced for three analysed strains, but only a single product for one strain. The sequence of spacer regions of Acetobacter bacteria were subjected to restriction analysis by HaeIII, HpaII and AluI enzymes. After analysis we obtained three different restriction groups of strains of A. pasteurianus for all enzymes. Amplified products were sequenced and nucleotide sequences were compared. The results showed two identical sequences for three strains A. pasteurianus 3612, A. pasteurianus 3614 and A. estunensis 3613. Intergenic region of A. pasteurianus 2374 is 40 % similar to other tested bacteria.