The study of the intra-specific phylogenetic structure of Leuconostoc oenos is essential to understand the participation of several strains in male-lactic fermentation (MLF). RFLP of the PCR-amplified 16S-23S rDNA intergenic spacer region (ISR) was performed in Leuc. oenos and other related species. The RFLP patterns with seven endonucleases were identical for the 37 Leuc. oenos strains, but differed from those obtained for all other species tested. This method could provide an invaluable insight for molecular identification of the wine leuconostocs. The RFLP relationships of members of the genera Leuconostoc and Weissella were highly similar to those previously reported by 16S and 23S rRNA sequencing studies. The 16S-23S rDNA ISR was sequenced in five strains of Leuc. oenos. A single tRNA(Ala) was detected. The ISR sequence seems to be identical in the two rRNA (rrn) operons found in Leuc. oenos and no significant sequence variation was observed between strains that revealed relative differences as previously shown by PFGE. Results from the present study demonstrated that Leuc. oenos is phylogenetically a very homogeneous species (according to DNA-DNA hybridization studies) and sustain that this species is different from the genus Leuconostoc. The extremely conserved ISR of these organisms suggests that Leuc. oenos strains currently isolated and characterized must have spread with the transfer of viticulture rather than coming from indigenous populations.