This paper evaluates the use of denaturing high performance liquid chromatography (DHPLC) technology for the discrimination of genetic differences in the 16S rRNA and alcohol dehydrogenase (AdhA) genes among bacterial species based on its efficiency and sensitivity to enable the detection and discrimination of different genetic sequences. In order to optimize DHPLC protocols for the analysis of 16S rRNA gene fragments amplified from bacteria, DNA isolated from 22 different strains representing main bacterial groups of interest in food microbiology was analyzed. While the use of 16S rRNA gene did not allow to difference two wild strains of Acetobacter malorum, this region revealed as useful to differentiate them from some pathogenic bacteria as Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Listeria innocua, Clostridium perfringens or Sthapylococcus aureus, from spoilage microorganisms as Xantomonas vesicatoria and Alicyclobacillus spp., and also from lactic acid bacteria as Lactobacillus plantarum, Lactobacillus casei, Lactobacillus sakei, Lactobacillus acidophilus, Streptococcus thermophilus and Lactococcus lactis that may suppose technological risk during vinegar production. The results demonstrate that 16S rRNA gene region is not adequate for the discrimination of the acetic acid bacteria (AAB) strains, so AdhA gene was selected to identify the two wild strains of Acetobacter malorum. Also 6 different reference strains of AAB were separated based on differences in AdhA gene region. DHPLC technology is able to discriminate between these two wild strains of A. malorum based on differences existing in the AdhA gene region. The data obtained indicate that the technique is capable of identifying most bacteria at species level and even at strain level with optimization of the protocols. This is of particular relevance in the case of AAB due to their poor recovery on culture media and difficulties in detection of viable but non cultivable cells.