Cannabis sativa is a multi-use plant, with industrial hemp varieties grown for food and fibre having low levels of the intoxicating phytocannabinoid (PC) Δ9-tetrahydrocannabinol (Δ9-THC). Despite significant breeding efforts to reduce PC levels, the risk of crops ‘going hot’, that is exceeding regulatory Δ9-THC thresholds, undermines market expansion of industrial hemp and restricts germplasm utilization. Blocking the synthesis of common PC precursors that initiate production and direct compound assembly is a valid approach that could be used to engineer PC-free plants or those with Δ9-THC concentrations below levels of detection. However, a comprehensive understanding of the pathways responsible for the fatty acid precursors of PCs is incomplete, which limits scope to develop knockout mutants or genotypes that lack the metabolic capacity to synthesize PCs. By examining the transcriptomes of C. sativa glandular trichomes that host the biosynthetic machinery for PC production, and probing gene expression across flower development, we identified several fatty acid pathway genes that are co-expressed with known PC pathway genes and show strong associations with PC content. Heterologous expression confirmed that the function of these genes is consistent with the hypothetical origin for PC fatty acid precursors, indicating that these gene products may represent key metabolic branch points in PC production. Removal or inactivation of these molecular targets in C. sativa could be used to further elucidate their involvement in PC biosynthesis and enable the development of novel PC-free varieties for industrial end-uses.