Lactobacillus plantarum is a species of considerable industrial and medical interest. To date, the lack of reliable molecular methods for definite identification at strain level has hindered studies of the population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in six housekeeping loci to determine the genetic relationship among isolates. The MLST system was established using 16 L. plantarum strains that were also characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rDNA intergenic spacer region (ISR). Ribotyping grouped the strains into four groups; however, RFLP analysis of the ISRs showed no differences in the strains analysed. In contrast, MLST had a good discriminatory ability. The sequence analysis of the six genes showed 14 different allelic combinations, with 12 of them represented by only one strain. By using this MLST approach we were able to confirm the identity of two strains deposited in the Spanish Type Culture Collection as different strains. Phylogenetic analysis indicated a panmictic population structure of L. plantarum and split decomposition analysis indicated that recombination plays a role in creating genetic heterogeneity in L. plantarum. As MLST allows precise identification, and easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be useful for the identification of valuable L. plantarum strains.