A two-step real-time SYBR Green I multiplex polymerase chain reaction (PCR) assay with melting curve analysis was developed for rapid detection of 19 Salmonella serotypes frequently encountered in humans, animals, and animal-associated meat products within the European Union. The first-step single-tube reaction (Multiplex PCR I), consisting of five primer pairs, classified an initial test panel of eight Salmonella serotypes into five groups on the basis of characteristic amplicon melting temperatures produced by each strain. Following designation into groups, two subsequent triplex reactions (Multiplex PCR II-G1 and II-G3) allowed for further identification of five Salmonella serotypes by their melting peak temperatures. Primers for serotype differentiation were designed to target the genes encoding either phase 1 and 2 flagellar antigens fliC and fljB or unique serotype-specific loci. In addition, the assay simultaneously screened for the presence of the ampicilin-amoxicillin, chloramphenicol-florfenicol, streptomycin-spectinomycin, sulfanomides, and tetracycline (ACSSuT)-type multidrug resistance pattern, indicated by the floR gene, and for the Salmonella virulence plasmid encoded by the svp operon in Salmonella serotype Typhimurium. The established multiplex assays were successfully tested on 97 isolates, comprising 37 distinct Salmonella serotypes and 12 non-Salmonella strains. The two-step assay correctly detected 19 of 37 Salmonella serotypes and all non-Salmonella strains produced negative results. Of the 19 serotypes detected in the assays, 7 serotypes, including Salmonella serotypes Ohio, Goldcoast, Livingstone, Kedougou, Enteritidis, Kentucky, ACSSuT-type Salmonella serotype Typhimurium DT104 and DT104b, as well as non-ACSSuT-type Salmonella serotype Typhimurium strains, were definitively identified. The developed multiplex real-time SYBR Green I PCR assay represents a more rapid and reliable method for identification of large numbers of Salmonella serotypes prevalent throughout the European Union than assays using phenotypic serotyping methods.