Abstract A fluorescent duplex-PCR test was developed based on polymorphisms of the THCA synthase gene in order to discriminate the fiber- and drug-type Cannabis sativa L. and to indicate the presence of Cannabis trace in suspected materials by the numbers and sizes of PCR-amplified products. DNA analysis of drug-type Cannabis resulted in two different PCR-amplified DNA fragments of 94 and 158 bp, whereas only the 94 bp PCR product was amplified from the fiber-type DNA. DNA test results of another 6 Cannabis sativa L. collected from the field agreed with chemotype determined by GC-MS. However, it was noted that the only intermediate drug-type sample tested gave a drug-type result for DNA testing. Specificity of the duplex PCR was shown by testing with DNA from species that may be related to Cannabis abuse, i.e., common hop ( Humulus lupulus L.), 2 narcotic plants ( Papaver somniferum and Mitragyna speciosa), tobacco ( Nicotiana tabacum) and human. Sensitivity of detection was as low as 100 pg of genomic DNA.