The Lactobacillus species by 16S Amplified Ribosomal DNA Restriction Analysis (16S-ARDRA) was identified. Lactobacilli are bacteria often found in foodstuffs of both animal and vegetable origins. On one hand, they play an important role in the food spoilage and, on the other hand, they are used as starter cultures in food fermentation processes. The species-specific identification by traditional biochemical methods is time consuming and not always fully effective. Therefore, more efficient techniques are searched for. We focused on rapid identification of Lactobacillus isolates from different habitats. Forty-nine collection strains and isolates belonging to the genus Lactobacillus were discriminated. ARDRA was carried out with two restriction endonucleases. For the comparison of similarity, the Jaccard coefficient and clustering by the unweighted pair group method with arithmetic averages (UPGMA) were used. The percentages of similarity between profiles varied from 22% to 100% (aluI) and from 27% to 100% (MspI). This method proved applicable to the differentiation of 10 species.