We developed a fluorogenic 5′ nuclease polymerase chain reaction (PCR) assay targeting the iap gene, which is highly conserved in all Listeria species. We used this assay to screen tissues obtained during a survey of cull sows. To date, we have detected Listeria species in in caecal contents (66%, 23 of 35 samples), tonsil homogenates (67%, 4 of 6 samples), and ileocaecal lymph nodes (75%, 6 of 8 samples), but infrequently in feces collected before slaughter (4.4%,1 of 23 samples). Taken together, these data indicate that second-generation 5′ nuclease PCR (TaqMan) assays accelerates detection and quantitation of Listeria during pork production. This may reduce product recalls as well as reduce human illness and deaths. The development of portable instruments for 5′ nuclease assays will expedite field surveys.