Oenococcus oeni is the organism of choice for promoting malolactic fermentation in wine. The population biology of O. oeni is poorly understood and remains unclear. For a better understanding of the mode of genetic variation within this species, we investigated by using multilocus sequence typing (MLST) with the gyrB, pgm, ddl, recP, and mleA genes the genetic diversity and genetic relationships among 18 O. oeni strains isolated in various years from wines of the United States, France, Germany, Spain, and Italy. These strains have also been characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCRamplified 16S-23S rRNA gene intergenic spacer region (ISR). Ribotyping grouped the strains into two groups; however, the RFLP analysis of the ISRs showed no differences in the strains analyzed. In contrast, MLST in oenococci had a good discriminatory ability, and we have found a higher genetic diversity than indicated by ribotyping analysis. All sequence types were represented by a single strain, and all the strains could be distinguished from each other because they had unique combinations of alleles. Strains assumed to be identical showed the same sequence type. Phylogenetic analyses indicated a panmictic population structure in O. oeni. Sequences were analyzed for evidence of recombination by split decomposition analysis and analysis of clustered polymorphisms. All results indicated that recombination plays a major role in creating the genetic heterogeneity of O. oeni. A low standardized index of association value indicated that the O. oeni genes analyzed are close to linkage equilibrium. This study constitutes the first step in the development of an MLST method for O. oeni and the first example of the application of MLST to a nonpathogenic food production bacteria.Oenococcus oeni, formerly Leuconostoc oenos (7), is the species of lactic acid bacteria (LAB) most frequently associated with malolactic fermentation (MLF) in wine. MLF, which occurs after alcoholic fermentation during wine making, is induced by the growth of LAB. Normally, spontaneous MLF takes place when LAB develop in wine after alcoholic fermentation. However, when fermentation by indigenous bacteria is relied upon, the fitness of the bacteria present to carry out MLF may be highly variable, and consequently their winemaking properties are unpredictable. In order to exercise greater control over wine-making processes, common winemaking practices therefore involve inoculation of wine with either commercially prepared strains or in-house winery strains of malolactic bacteria (4). Therefore, the differentiation of O. oeni strains at the strain level becomes a major concern, since their adaptation to wine and influence on organoleptic quality are strain specific (2). Moreover, manufacturers of malolactic starters need accurate control of their products, and winemakers have to be able to recognize the inoculated strains during vinification. The need for positive identification of different isolates is also acknowled...