Aims: To develop a 24-h system for the detection of Listeria monocytogenes in ham. Methods and Results: An immunomagnetic separation (IMS) of bacteria directly from ham followed by extraction of DNA and detection using a new multiplex polymerase chain reaction (PCR) was used. The PCR method used one primer pair targeted at the listeriolysin O gene of L. monocytogenes and the other pair for a region of the 23S rRNA genes of Listeria, giving products of 706 and 239 bp, respectively. The combined IMS/PCR was calculated to be capable of detecting as few as 1-1 L. monocytogenes cells g(-1) in a 25-g ham sample. Conclusions: The process produced acceptable results, but the IMS step is the main barrier to further improvement of sensitivity. The DNA isolation was the most time-consuming step in the process. Significance and Impact of the Study: A 24-h test for the presence of L. monocytogenes will be useful to the food industry and significantly assist in the timely investigation of outbreaks.