Salmonella enterica serovar Indiana ( S. Indiana) is a newly emerging pathogen with high levels of drug resistance. It has become one of the most common Salmonella serovars in China with a worldwide distribution, posing significant public health concerns. Detection of S . Indiana by traditional bacteriological methods is time-consuming and laborious, which prevents timely surveillance and effective control of the pathogen. In this study, comparative genomics was used to identify an A7P63_13850 gene that is uniquely present in S . Indiana, but not in other Salmonella serovars or any non- Salmonella bacteria. Then, a polymerase chain reaction (PCR) assay targeting this serovar-specific gene was established for specific detection of S . Indiana. The detection limit of this method is 10 pg per reaction for bacterial genomic DNA, being equivalent to 100 colony-forming units (CFU) per reaction. The established PCR amplifies all S . Indiana strains ( n = 56), but none of other Salmonella serovars ( n = 146) and non- Salmonella species ( n = 14). The assay established in this study was also used to detect clinical samples from poultry, showed a positivity of 14.7% (23/156) for S . Indiana, which were verified by bacteriological methods. The highly sensitive and serovar-specific PCR for S . Indiana established in this study is suitable and convenient for detection of S . Indiana which aids in surveillance and control of the pathogen.