Listeria monocytogenes is an opportunistic foodborne pathogen that is a serious health hazard worldwide. In this study, a polymerase chain reaction (PCR) assay was developed for the detection of L. monocytogenes using a novel species-specific target sequence (a region of lmo0754gene) identified by a comparative genomic approach. An internal amplification control was incorporated into this PCR system. The assay allowed amplification of a 331-bp fragment only from the genomic DNA of L. monocytogenes strains and not from other Listeria species, as well as some non-Listeria species. The detection limit of the PCR assay was 55 copies/PCR with L. monocytogenes genomic DNA. Applying this PCR assay to artificially contaminated milk samples, low levels of L. monocytogenes (1–10 cfu/mL of milk) were detected after 6–9 h incubation in selective culture enrichment (UVM1).