More than 2,500 serovars of Salmonella species have been reported to date. A multiplex-PCR method was developed and evaluated for discriminating the four Salmonella enterica subsp enterica serovars, namely, S. Enteritidis, S. Typhimurium, S. Thompson and S. Infantis, most commonly isolated in Japan. Twenty-two serovars of 84 Salmonella strains and 7 species of non-Salmonella strains were evaluated using primer pairs specific for the detection of Salmonella spp. Multiplex PCR generated, with 100% specificity, the expected amplicon of 333, 413, 551 and 658 bp of S. Enteritidis, S. Infantis, S. Typhimurium, and S. Thompson, respectively, while an additional non-specific amplicon (about 1,000 bp) was observed for S. Infantis, but it had no practical impact in the bacterial detection. This multiplex PCR assay can be applied to identify and discriminate clinically significant strains of Salmonella serovars rapidly and accurately without the need for serological examination.