Salmonella is a major food-borne pathogen in humans and a cause of local or systemic disease in animals. Therefore, rapid and reliable methods to detect these poultry-associated Salmonella serotypes are necessary for efficient control of Salmonella in poultry. The present study aimed to develop a real-time multiplex PCR (MqPCR) method to simultaneously detect and/or differentiate Salmonella sp. and poultry-associated serotypes, including Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Pullorum, and Salmonella Gallinarum. A MqPCR method was designed using four specific primer pairs and probes for the detection of Salmonella sp., including S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum. Additionally, a novel TaqMan-based MqPCR method combined with propidium monoazide (PMA) treatment was developed for the simultaneous quantification of viable cells of Salmonella sp. and these four Salmonella serotypes in rinse water of chicken carcasses. The MqPCR assay specifically detected Salmonella sp., S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum, showing 100% sensitivity and 100% specificity. This optimized PMA-MqPCR assay could detect live Salmonella (10(0)-10(6) CFU/reaction) without enrichment in live/dead cell mixtures from spiked rinse water of chicken carcasses. The procedure for detecting live Salmonella required less than 2 h to complete. This PMA TaqMan-based MqPCR technique facilitates accurate and rapid monitoring of contamination with viable Salmonella. Also, the assay enables simultaneous identification of S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum in rinse water of chicken carcasses. The assay developed in this study will be useful in diagnostic laboratories for improving Salmonella control in poultry and poultry products.