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The objective was to study the genetic structure and degree of variability of hemp (Cannabis sativa L.) varieties. Six varieties of hemp were analyzed by random amplified polymorphic DNA (RAPD) analysis, using 10 plants per variety. The varieties were a dioecious landrace, a dioecious selection from it, a cross-bred cultivar, a monoecious variety, a drug strain, and an inbred female line. The genetic complexity of each cultivar was investigated by determining the number of bands produced by the primers used, the number of fixed and polymorphic loci, the average allele frequency, and the heterozygosity. A good correlation was found between these parameters and the genetic origin and breeding strategy of each variety. The average polymorphism over all varieties and loci was 97.1%; the single cultivar polymorphism ranged from 31.1 to 85.5%. Heterozygosity ranged from 0.05 (inbred female line) to 0.26 (cross-bred Fibranova). The average heterozygosity calculated over all 102 loci and all plants studied was 0.29. The Fst (Wright's fixation index) value calculated for all loci was 0.48, and only 33.3% of the scored loci had higher values and can be considered informative for cultivar identification. A Fisher's test based on allele frequencies suggested complete differentiation among all varieties, with the exception of the Italian dioecious varieties Carmagnola and CS, for which no discriminating alleles were found. The correlations among the molecular data and the genetic structure of the different cultivars and the consequences in relation to variety discrimination in hemp are discussed., HEMP is a traditional crop in Europe and Asia. It belongs to the Cannabaceae family, which comprises two genera: Cannabis and Humulus (hops). Despite hemp's past importance for both the [...]