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Abstract Cannabichromene (CBC) is a nonpsychoactive cannabinoid with pharmaceutical potential that is attracting significant interest for its potential therapeutic applications. However, the factors governing CBC production in Cannabis sativa remain largely unknown. This study aimed to investigate the variation in CBC accumulation within different Type III (cannabidiol dominant) C. sativa cultivars. We hypothesized that distinct subpopulations with differing CBC production exist within these cultivars. Quantitative analysis was performed on a diverse collection of Type III C. sativa cultivars to determine CBC levels. We assessed CBC accumulation across generations and environments for select accessions. Temporal changes in CBC during flowering and its distribution in floral and leaf tissues were also examined. Our analysis revealed distinct chemotypic subpopulations exhibiting varying CBC production levels. Notably, certain cultivars consistently displayed high CBC levels across generations and diverse growing conditions, suggesting a strong genetic influence. The observed intra‐varietal variation and the stability of high‐CBC phenotypes indicate a significant genetic component in CBC biosynthesis. Understanding this complex interplay of genetic and environmental factors has important implications for breeding programs focused on enhancing CBC production in C. sativa .