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Genetic markers can be used in seeds and in plants to distinguish drug-type from fiber-type <i>Cannabis Sativa</i> L. varieties even at early stages, including pre-germination when cannabinoids are not accumulated yet. With this aim, this paper reports sequencing results for <i>tetrahydrocannabinolic acid synthase</i> (<i>THCAS</i>) and <i>cannabidiolic acid synthase</i> (<i>CBDAS</i>) genes from 21 <i>C. sativa</i> L. varieties. Taking into account that <i>THCAS</i>- and <i>CBDAS</i>-derived enzymes compete for the same substrate, the novelty of this work relies in the identification of markers based on both <i>THCAS</i> and <i>CBDAS</i> rather than <i>THCAS</i> alone. Notably, in our panel, we achieved an adequate degree of discrimination (AUC 100%) between drug-type and fiber-type cannabis samples. Our sequencing approach allowed identifying multiple genetic markers (single-nucleotide polymorphisms&#8212;SNPs&#8212;and a deletion/insertion) that effectively discriminate between the two subgroups of cannabis, namely fiber type vs. drug type. We identified four functional SNPs that are likely to induce decreased <i>THCAS</i> activity in the fiber-type cannabis plants. We also report the finding on a deletion in the <i>CBDAS</i> gene sequence that produces a truncated protein, possibly resulting in loss of function of the enzyme in the drug-type varieties. Chemical analyses for the actual concentration of cannabinoids confirmed the identification of drug-type rather than fiber-type genotypes. Genetic markers permit an early identification process for forensic applications while simplifying the procedures related to detection of therapeutic or industrial hemp.