In many countries, including Japan, cultivation of Cannabis sativa ( C. sativa ) for drug use has been illegal and prohibited. Recently, seeds for cultivation purposes are easily available from internet shops, and then we have often been requested to identify C. sativa . The identification has conventionally been performed by morphological and chemical tests. But, it can be difficult to identify tiny and fragmenting samples as C. sativa even if these tests are performed.   In this study, we aimed to establish a method based on DNA analysis. As an initial step, we attempted a method reported by Linacre et al, however, cross-amplification between C. sativa and Humulus lupulus ( H. lupulus ) with C. sativa specific primers (G and H) was observed. To avoid this cross-amplification, we designed a new primer specific for C . sativa (cp-Can) on trnL intron of chloroplast DNA. DNA samples from nine plants including C. sativa and H. lupulus were amplified using the green plant universal primer pair and the cp-Can. After subsequent agarose gel electrophoresis, C. sativa DNA showed two bands, whereas the other plant DNA showed one band, indicating the clear distinction from the other plants tested. In addition, a BLAST search with the cp-Can sequences showed no cross-activity with other plants. The present method is very simple, rapid, sensitive, and useful for the identification of C. sativa .