Abstract: ObjectiveReasonable sampling size for wild Cannabis sativa L. was analyzed and determined to provide reference for comprehensive understanding of genetic diversity of domestic C. sativa L. resources and formulating protec-tion strategies for it. MethodNinety-six representative individuals of wild C. sativa L. population from Horqin sandy land were randomly selected. Using computer simulation method, each sampling group with different individuals ( 5 , 10 , 15,20,25,30,35,40,50,60,70,80,90,96) were randomly sampled 20 times. ISSR molecular marker method was carried out to estimate the genetic diversity and determine the proper sampling size. ResultPCR amplification was conducted on 96 sampled C. sativa L. individuals by 9 primers screened out of 60 ISSR primers. The results showed that 76 loci with 48 polymorphic loci were identified, and polymorphic rate was 63.16%. Observed average alleles(Na) number was 1.632, effective alleles(Ne) number was 1.250, Nei's gene diversity index(H) was 0.156,and Shannon's information index(I) was 0.245. Fitted curves of different genetic parameters indicated that the genetic diversity increased sharply for the first four sampling groups (5,10,15 and 20 individuals respectively ). As sampling size enlarged, the increasing trend of genetic diversity was slowed. When the sampling size was over 25 individuals, Na,Ne,H and I contained over 90% of genetic diversity of wild C. sativa L population. ConclusionThe sampling size should be no less than 25 indi-viduals when using ISSR molecular marker to evaluate genetic diversity of C. sativa L.,as it can reflect the genetic di-versity of the population.