Veillonella atypica, Veillonella dispar and Veillonella parvula cannot be reliably distinguished by conventional phenotypic tests, including the API ZYM test. In this study, restricted fragment-length polymorphism (RFLP) analysis of 16S rDNA amplified by polymerase chain reaction (PCR) was used to generate restriction profiles of the type strains of V. atypica, V. dispar and V. parvula and 20 Veillonella strains isolated from oral sites. 16S rRNA gene sequences from isolated genomic DNA samples were amplified by PCR. PCR products were purl fled and characterized by single digestion with 13 restriction endonucleases. Among them, MnlI was found to discriminate the respective reference strains, and the clinical isolates were assigned to one of the three species on the basis of their restriction profiles by digestion with MnlI. Thus, RFLP analysis of PCR-amplified 16S rDNA, using MnlI, is a rapid and reliable method for the differentiation of V. atypica, V. dispar and V. parvula.