A multiplex polymerase chain reaction (PCR) assay has been developed for rapid and reliable identification of Leuconostoc species, by using species-specific primers targeted to the genes encoding 16S rRNA. This assay can detect and differentiate Leuconostoc species from mixed populations in natural sources as well as from pure cultures, within 3 h. This assay system consists of a total of 10 primers, two primers from each target species, and comprises two multiplex PCR reactions: one reaction for Leuconostoc carnosum, Leuconostoc citreum and Leuconostoc mesenteroides, and another reaction for Leuconostoc gelidum and Leuconostoc lactis. This multiplex PCR assay was used to identify 31 Leuconostoc strains isolated from kimchi, a fermented-cabbage product, and the results showed perfect correlation with the results of a polyphasic method, including 16S rDNA sequencing and DNA-DNA hybridization. In addition, this assay enables simultaneous detection of the above-mentioned Leuconostoc species when chromosomal DNA from these Leuconostoc species was mixed. Thus, these results suggest that this multiplex PCR is a rapid and reliable method for identification of Leuconostoc species in pure cultures or in mixed populations.