Cannabinoids are specific secondary metabolites in the plant of Cannabis sativa L. Tetrahydrocannabinol (THC）and cannabidiol (CBD）are the main constituents of cannabinoids. To distinguish the chemotype and genotype of cannabinoids of Cannabis germplasm, 23 Cannabis germplasm resources (including 69 single plants) from different areas of China were selected to analyze content characteristics of THC and CBD, and also gene polymorphism of key enzymes during the pathway of cannabinoid biosynthesis. The results showed that the cannabinoid contents of 69 single plants were significantly different. The average content of THC was 0.56% ranging from 0.01% to 2.45%, The average content of CBD was 0.53% ranging from 0 to 2.24%. According to CBD/THC ratio, all Cannabis plants can be divided into 3 chemotypes: drug-type (accounting for 44.93%), intermediate-type (accounting for 20.29%) and fiber-type (accounting for 34.78%). The THC content of these plants, including 93.5 % plants among drug-type and 71.4% plants among intermediate-type, are more than 0.3%. The THC content of plants in fiber-type are less than 0.08%. Genotypes in drug-type, intermediate-type and fiber-type were BT/BT, BT/BD and BD/BD respectively. The sequences alignment showed that BT allele (THCAS) had 10 mutation sites in nucleotide sequences, and 4 mutation sites in the deduced amino acids. The BD allele (CBDAS) had 4 mutation sites in nucleotide sequences, but no mutation in the deduced amino acids. According to the polymorphism of THCAS and CBDAS, a codominant multiplex PCR molecular marker was designedto identify three chemotypes of cannabinoids. This study revealed that the relationship among cannabinoid contents, chemotypes and genotypes, which can provide a theoretical foundation for genetic research and utilization of cannabinoids.