Abstract: Objective To establish a polymerase chain reaction assay for the rapid detection of Salmonella Anatum and evaluate its specificity,sensitivity and detection limit. Methods The genomes of Salmonella spp. isolated before August 2015 were downloaded from GenBank database to find certain specific genes which are only present in S. Anatum strains. Primers targeting the specific genes of S. Anatum were designed and the PCR assay was established. The specificity, sensitivity and the detection limit of the assay were evaluated by using pure cultured bacterial strains,clinical and simulated stool samples respectively. Results The PCR assay for the rapid detection of S. Anatum established based on a novel marker gene AW58_15605 in this study had positive amplification of the specific gene for all the S. Anatum strains,including pure cultured bacterial strains and clinical samples,whereas no amplifications were observed for other serotypes of Salmonella and other enteric pathogens causing diarrheal diseases. The specificity and sensitivity of the assay were all 100%. The detection limit of the PCR assay for S. Anatum with purified DNA as template reached 59.1 cfu/g in simulated stool samples after overnight selective enrichment,which was 105 fold higher than that before enrichment. Conclusion The established PCR assay for the rapid detection of S. Anatum based on a novel gene could identify S. Anatum with high specificity and sensitivity. Therefore,it has a good potential for the application in S. Anatum infection clinical diagnosis and outbreak response.