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Panax notoginseng is a traditional Chinese medicinal herb. With its wide applications in medicine, food products, health care, cosmetics and other industries, identification and detection of P. notoginseng have become critical. In this study, we developed a real-time polymerase chain reaction (qPCR) assay with specific and internal control probes based on chloroplast psbA-trnH gene and 18S rDNA to identify and detect P. notoginseng in different products. A specific primer and probe set was designed by comparing psbA-trnH sequence among P. notoginseng, Panax ginseng, Panax quinquefolius and Panax japonicus. Besides, the 18S rDNA gene was used as an internal control. The qPCR condition was optimized with the sensitivity to be 0.005 ng/μL and high specificity. There was no cross-reaction among 18 closely related common Chinese herbs. Detection results of 28 commercially available P. notoginseng products were consistent with the standardized sequencing results. This study provides a rapid, specific and sensitive assay for detection of P. notoginseng in different products.