Salmonellaspp. can cause animal and human salmonellosis. In this study, we established a simple method to detect allSalmonellaspecies by amplifying a specific region within theflgEgene encoding the flagellar hook protein. Our preliminary sequence analysis among flagella-associated genes ofSalmonellarevealed that althoughSalmonellaGallinarum andSalmonellaPullorum are lacking flagella, they did have flagella-associated genes, includingflgE. To investigate in detail, a comparativeflgEsequence analysis was conducted using different bacterial strains including flagellated and non-flagellated Salmonella as well as non-Salmonellastrains. Two unique regions (481-529 bp and 721-775 bp of the reference sequence) within theflgEopen reading frame were found to be highly conserved and specific to allSalmonellaspecies. Next, we designed a pair of PCR primers (flgE-UP andflgE-LO) targeting the above two regions, and performed aflgE-tailored PCR using as template DNA prepared from a total of 76 bacterial strains (31 flagellatedSalmonellastrains, 26 non-flagellatedSalmonellastrains, and 19 other non-Salmonellabacteria strains). Results showed that specific positive bands with expected size were obtained from allSalmonella(including flagellated and non-flagellatedSalmonella) strains, while no specific product was generated from non-Salmonellabacterial strains. PCR products from the positive bands were confirmed by DNA sequencing. The minimum detection amount for genomic DNA and bacteria cells reached 18.3 pg/mu L and 100 colony-forming unit (CFU) per PCR reaction, respectively. Using theflgE-PCR method to detectSalmonellain artificially contaminated milk samples, as low as 1 CFU/mLSalmonellawas detectable after an 8-h pre-culture. Meanwhile, theflgE-tailored PCR method was applied to evaluate 247 clinical samples infected withSalmonellafrom different chicken breeding farms. The detection results indicated thatflgE-PCR could be used to specifically detectSalmonellain concordance with the traditional bacterial culture-based detection method. It is worthwhile noticed that identification results usingflgE-tailored PCR should be completed within less than 1 day, expanding the result of much faster than the standard method, which took more than 5 days. Overall, theflgE-tailored PCR method can specifically detect flagellated and non-flagellatedSalmonellaand can serve as a powerful tool for rapid, simple, and sensitive detection ofSalmonellaspecies.