Technical Abstract: Transcriptional regulator genes encode a group of specialized molecules that play essential roles in microbial responses to changing external conditions. These genes have been shown to possess species or group specificity and are useful as detection targets for diagnostic application. The present study examined transcriptional regulator genes from a nonpathogenic species Listeria welshimeri that often causes difficulty in the laboratory diagnosis of Listeria monocytogenes-related foodborne disease. The comparison of transcriptional regulator gene sequences from L. welshimeri Special Listeria Culture Collection 5334 genome with other microbial genes at GenBank led to the identification of lwe1801. The application of polymerase chain reaction (PCR) primers derived from lwe1801 enabled the amplification of a specific band of 510 bp from the DNA of L. welshimeri only and not from that of other Listeria and common bacterial species. Given its unique presence in L. welshimeri, the transcriptional regulator gene lwe1801 constitutes a novel molecular marker for species-specific confirmation of this bacterium.