Salmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping of Salmonella enterica isolates based on specific single-nucleotide polymorphisms in fragments of fljB, gyrB, and ycfQ. Simultaneous gene scanning of fljB, gyrB, and ycfQ by high-resolution melting-curve analysis of 417 Salmonella isolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases from Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Dublin, in all cases from Salmonella enterica serotype Ohio and Salmonella enterica serotype Rissen, from Salmonella enterica serotype Mbandaka and Salmonella enterica serotype Kentucky, and from Salmonella enterica serotype Bredeney, Salmonella enterica serotype Give, and Salmonella enterica serotype Schwarzengrund. To differentiate the most frequent Salmonella serotype, Enteritidis, from some S. Dublin isolates, an additional single PCR assay was developed for specific identification of S. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with an S. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39 Salmonella serotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhuman Salmonella isolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.