Cannabis is an annual herb and a versatile， sustainable crop， while uncovering the population diversity remain poorly investigated. In this study， the EST-SSR molecular markers were used to analyze the genetic diversity and population structure of cannabis. The results showed that a total of 113 score bands were amplified using 20 pairs of primers， of which all （100%） were polymorphic. A total of 232 alleles were detected， with an average of 4.0176 alleles per primer pair. The average observed heterozygosity （Ho） was 0.7102， and the average expected heterozygosity （He） was 0.6935. The Shannon information index of 200 individuals ranged from 0.7204 - 2.4625， with an average value of 1.5368. Polymorphism information content （PIC） ranged from 0.3519 to 0.8801， with an average of 0.6558. The mean gene flow （Nm） was 13.6525.Based on population genetic structure， principal component analysis and unweighted group analysis （UPGMA） with arithmetic mean， cannabis materials were grouped into 3 groups.. The results were similar between the clustering methods， but the distribution of minority individual plants was different among the three models. The results of clustering， genetic diversity and genetic similarity coefficient showed that cannabis individuals were closely related to each other. In addition， five pairs of core primers， which were able to distinguish the test germplasm， were deployed for the fingerprint construction. Collectively， these results provided a reference for the future breeding， genetic improvement and collection of core germplasm resources of cannabis.