Taxonomic differentiation between Lactococcus sp. and Leuconostoc sp. can sometimes be misleading due to the morphological and biochemical similarities between both genera. Therefore, several molecular techniques have been applied to identify these bacteria. Restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal RNA gene was used to generate restriction profiles of 9 strains of Lactococcus sp. and 5 of Leuconostoc sp. This method utilizes a set of universal primers for amplification of the 16S rRNA region of typical lactic acid bacteria species. The size of the amplified products was about 1500 bp and the amplicons of the different species could be differentiated from each other with four restriction endonucleases: TaqI, EcoRI, BamHI and HindIII. These restriction enzymes were selected based on nucleotide sequences of 16S rRNA genes for LAB available in databases. Our study demonstrates that DNA of 16S rRNA from strains of Lactococcus sp. contains single restriction site for EcoRI and two restriction sites for TaqI enzymes, 16S rRNA DNA from strains of Leuconostoc sp. contains a single restriction site for each enzyme (HindIII, BamHI) and four restriction sites for TaqI. This result is in good agreement with analysis in silico of 16S rRNA genes published in the National Center for Biotechnology Information (NCBI). These findings led to modify the classification obtained by biochemical methods for five examined strains of lactic acid bacteria. In summary, our study demonstrated that the RFLP analysis applied is a useful method for rapid differentiation between Lactococcus sp. and Leuconostoc sp.