Aim: To develop a novel multiplex polymerase chain reaction (PCR) assay with six primer pairs for Salmonella subspecies identification. Methods and Results: Five primer pairs were chosen to detect the genes (fljB, mdcA, gatD, stn and STM4057) responsible for several phenotypic traits or encoding (sub) species-specific regions. A primer pair for invA was added to simultaneously detect Salmonella. The combination of these primer pairs was expected to give unique results to all subspecies, including Salmonella bongori. The multiplex PCR assay was optimized and evaluated with 53 Salmonella strains representing all S. enterica subspecies, S. bongori and five non-Salmonella strains. The multiplex PCR assay revealed that the genotypes were well correlated with the phenotypes in the Salmonella strains tested. The unique band patterns to their subspecies were generated from 94 center dot 3% (50/53) of the Salmonella strains, and no product from other strains by the multiplex PCR assay. Conclusions: The multiplex PCR assay we developed was found to be a rapid, specific and easy to perform method compared with traditional biochemical tests for Salmonella subspecies identification, especially for rapid screening of large numbers of samples. Significance and Impact of the Study: The assay will be useful for characterizing Salmonella isolates from reptiles, which belong to various subspecies, and therefore add to the scientific understanding of reptile-associated Salmonellosis.