Background: Salmonella enterica serovar Typhi, as causative agent of typhoid fever, is one of the most important endemic pathogens. Non-typhoidal Salmonella serovars, including Typhimurium, Infantis, and Enteritidis are amongst the most prevalent serotypes worldwide and in developing areas such as Iran. The aim of this study was to apply a uniplex PCR for rapid detection of Salmonella spp., and a multiplex PCR for the simultaneous detection of the four most common Salmonella serovars in Iran. Methods: Current research was done in 2010 at Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. For detection of Salmonella spp a pair of primers was used to replicate a chromosomal sequence. Four other sets of primers were also designed to amplify the target genes of four Salmonella species including S. typhi, and three non-typhoidal Salmonella spp (S. enteritidis, S. infantis, and S. typhimurium). The assay specificity was investigated by testing 15 different Salmonella serovars and 8 other additional non-Salmonella species. Results: The Salmonella genus-specific PCR yielded the expected DNA band of 404 bp in all Salmonella spp., strains tested. The uniplex and multiplex PCR assays produced also the expected fragments of 489 bp, 304 bp, 224 bp, and 104 bp for serovars Typhi, Enteritidis, Typhimurium, and Infantis, respectively. Each species-specific primer pair set did not show any cross-reactivity when tested on other Salmonella serovars or other non- but related- Salmonella strains. Conclusion: Both uniplex and multiplex PCR protocols had a good specificity. They can provide an important tool for the rapid and simultaneous detection and differentiation of the four most prevalent Salmonella serovars in Iran.