Cannabis has been cultivated for millennia with distinct cultivars providing either fiber and grain or tetrahydrocannabinol. Recent demand for cannabidiol rather than 20 tetrahydrocannabinol has favored the breeding of admixed cultivars with extremely high cannabidiol content. Despite several draft Cannabis genomes, the genomic structure of cannabinoid synthase loci has remained elusive. A genetic map derived from a tetrahydrocannabinol/cannabidiol segregating population and a complete chromosome assembly from a high-cannabidiol cultivar together resolve the linkage 25 of cannabidiolic and tetrahydrocannabinolic acid synthase gene clusters which are associated with transposable elements. High-cannabidiol cultivars appear to have been generated by integrating hemp-type cannabidiolic acid synthase gene clusters into a background of marijuana-type cannabis. Quantitative trait locus mapping suggests that overall drug potency, however, is associated with other genomic regions needing 30 additional study. Resources available online at: http://cannabisgenome.org SummaryA complete chromosome assembly and an ultra-high-density linkage map together identify the genetic mechanism responsible for the ratio of tetrahydrocannabinol 35 (THC) to cannabidiol (CBD) in Cannabis cultivars, allowing paradigms for the evolution and inheritance of drug potency to be evaluated. 45 has also frustrated attempts to assemble complete chromosomes from thousands of Veronica Tonnell contributed DNA isolation for the mapping population. We thank