Salmonellainfections continue to cause gastrointestinal and systemic disease throughout the world.Salmonella typhimuriumDT104 further poses a major health concern due to its acquisition of resistance to multiple antibiotics. The rapid detection of multiresistantS. typhimuriumDT104 would facilitate strategies aimed at controlling this pathogen. We developed a specific and sensitive polymerase chain reaction (PCR) assay that amplifies a segment of DNA that is conserved in multiresistantS. typhimuriumDT104. To provide further specificity for this PCR-based diagnostic test, we amplified two other gene fragments that are present inS. typhimuriumDT104. A multiplex PCR containing primers for targeted sequences resulted in the amplification of predicted size fragments fromS. typhimuriumDT104 exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline) or ASSuT resistance phenotypes. A minor modification of the multiplex PCR enabled the detection of other related multiresistantSalmonellasuch asS. typhimuriumU302. To augment the detection process, we also designed a fluorogenic PCR assay that can detect the DNA of multiresistantS. typhimuriumDT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistantS. typhimuriumDT104, or potentially the next emerging multiresistantSalmonella, can be accurately detected in only 3–4 h.