The tuf gene, which encodes the elongation factor Tu, is universally distributed in Gram-positive bacteria. A small region of the tuf gene was evaluated for the differentiation of Lactobacillus strains using restriction fragment length polymorphism (RFLP) analysis. The 803 bp portion of tuf genes from 9 closely-related Lactobacillus reference strains were successfully amplified by polymerase chain reaction (PCR), and restriction patterns of each were evaluated with a set of restriction enzymes, which included AluI, HaeIII, and Sau3AI. The results implied that restriction patterns obtained from 2 of these restriction enzymes (AluI and HaeIII) could effectively differentiate closely-related Lactobacillus species.