The development and validation of a multiplex PCR assay for the detection of Listeria that can be employed in routine investigation of food samples are described. The assay, which employs a short culture enrichment step followed by isolation of bacterial cells and detection by multiplex PCR reaction, is highly sensitive and specific for the detection of Listeria monocytogenes and all other Listeria species. Over 350 food samples were tested in parallel by standard cultural procedures and the PCR assay, with no false-positive or false-negative results obtained with the PCR assay. Compared to the standard cultural methods the PCR assay is highly sensitive, cost effective and extremely rapid with results obtained within 48 h from sample receipt.