Salmonella enterica is a significant cause of gastroenteritis worldwide, with serovars Typhimurium and Heidelberg being particularly prevalent, which have broad host ranges infecting poultry, dairy animals, and humans. Traditional methods used for the detection of Salmonella from contaminated food products are time-consuming and labor-intensive. The aim of this study was to develop a sensitive and rapid PCR-based detection method with optimized specificity for high-throughput screening of food and clinical samples. We used bioinformatics to identify potential serovar-specific regions from the available S. enterica sequenced genomes. We designed primer pairs to targeted regions unique to Typhimurium and Heidelberg. A primer pair targeting a putative cytoplasmic protein STM4492 amplified a 759-bp product specific to Typhimurium, and a primer pair targeting a putative inner membrane protein STM2745 amplified a 199-bp product from both Typhimurium and Heidelberg. A primer pair for the oriC locus was used to identify all Salmonella. We screened 217 isolates including the Salmonella reference collections A and B, validating the specificity of each primer set. Next, a multiplex PCR (mPCR) assay and quantitative real-time PCR assay were optimized for identification and differentiation of Typhimurium and Heidelberg. An mPCR assay was developed and successfully detected S. enterica isolates from inoculated Cheddar cheese, raw turkey, and cooked turkey at concentrations as low as 1 CFU/g of food. The reaction conditions for this mPCR have significantly reduced the time needed to identify S. enterica Typhimurium and Heidelberg, making this a rapid selective tool.