Two closely related lactic acid bacteria, Lactobacillus sakei and Lactobacillus curvatus, are very difficult to be rapidly differentiated. Here we report multiplex polymerase chain reaction (PCR)-based restriction enzyme analysis that is useful for rapid and reliable identification of these two species. This method employs both polymerase chain reaction (PCR) and restriction enzyme analysis (REA). First, multiplex-PCR using three primers that were designed from 16S rDNA sequence produces two bands, a 433-bp and a 623-bp band. A 433-bp band represents only L. sakei and L. curvatus among lactobacilli and genetically related bacteria, and a 623-bp band is used for further identification by restriction analysis. Second, restriction analysis of 623-bp band using Hind III restriction enzyme discriminates L. sakei from L. curvatus. This method could identify 28 strains as L. sakei or L. curvatus, which were frequently isolated from kimchi, a traditional fermented cabbage product in South Korea. Therefore, these results suggest that this method is simple, rapid, and reliable for the identification of L. sakei and L. curvatus species.