6 target genes specific for Listeria spp. were mined and identified by genomic approach. The multiplex PCR assay showed good specificity and sensitivity. There is no interference on detection of Listeria spp. with other pathogens at high concentration. The multiplex PCR assay has improved the efficiency of detection of Listeria spp. in animal derived food products. Detection in 200 animal-derived foods was consistent with standard methods.