Abstract: Contamination of lettuce by Salmonella has caused serious public health problems. Polymerase chain reaction (PCR) allows rapid detection of pathogenic bacteria in food, but it is inaccurate as it might amplify DNA from dead target cells as well. This study aimed to investigate the stability of DNA of dead Salmonella cells in lettuce and to develop an approach to detecting viable Salmonella in lettuce. Salmonella-free lettuce was inoculated with heat-killed Salmonella Typhimurium cells and stored at 4 °C. Bacterial DNA extracted from the sample was amplified by real-time PCR targeting the invA gene. Our results indicate that DNA from the dead cells remained stable in lettuce for at least 8 d. To overcome this limitation, propidium monoazide (PMA), a dye that can selectively penetrate dead bacterial cells and cross-link their DNA upon light exposure, was combined with real-time PCR. Lettuce samples inoculated with different levels of dead or viable S. Typhimurium cells were treated or untreated with PMA before DNA extraction. Real-time PCR suggests that PMA treatment effectively prevented PCR amplification from as high as 108 CFU/g dead S. Typhimurium cells in lettuce. The PMA real-time PCR assay could detect viable Salmonella at as low as 102 CFU/mL in pure culture and 103 CFU/g in lettuce. With 12-h enrichment, S. Typhimurium of 101 CFU/g in lettuce was detectable. In conclusion, the PMA real-time PCR assay provides an alternative to real-time PCR assay for accurate detection of Salmonella in food.