Aconite species (subgenus Aconitum) are one of the most toxic plants in Japan. Cases of aconite poisoning caused by unintentional consumption of the plant are not rare in Japan, and aconitine, the primary toxic compound in aconite, has been also used to commit suicides and murders. In general, the presence of aconite poisoning is confirmed based on whether aconitine or related alkaloids are detected in the blood or body fluids of the victim; however, such evidence related to aconite itself has not been investigated in detail. Recently, the development of an identification method focused on the ITS region of several Japanese toxic plants by real-time PCR was reported [1]. We have been investigating DNA from Japanese aconite [2] and [3], and wanted to develop a similar new method. The aim of this study was to identify the presence of Japanese aconite using multiplex PCR. In addition, the sequence diversity leading to the aconite individuality was also investigated.