AbstractCannabis sativa played a pivotal role across different industries. Recently, industrial hemp, particularly in the case of Cannabidiol (CBD), gained attention for its therapeutic potential. This study evaluated cannabinoid variability and genetic diversity within 43 industrial hemp individuals, primarily Turkish, across various plant parts and growth stages, and inflorescences of females showed significant CBD content. The highest contents were observed in Turkish landraces (0.55–8.05% with an average of 3.26%), making them valuable genetic resources for high CBD. Genetic structure revealed distinct populations based on gender and influenced by geographical origin. Analysis of Molecular Variance showed 92% of genetic variation observed within populations and indicated a promising source of novel allelic diversity in the Turkish gene pool. Turkish females showed significant genetic diversity [No. of Different Alleles (Na) 1.507, No. of Effective Alleles (Ne) 1.226, Shannon’s Information Index (I) 0.258, and Percentage of Polymorphic Loc (%P) 74.91], exhibiting richer genetic variation than their international peers. Principal Coordinate Analysis unveiled gender-specific genetic differences, and admixture clusters shed light on genetic interactions and historical connections among diverse populations. Unweighted Pair-Group Method with Arithmetic Averaging highlighted unique genetic profiles and distinct genetic lineages. Genome-wide association study revealed a highly significant male-specific genetic marker explained 50% of the phenotypic variation. These findings inform future breeding strategies and conservation efforts and contribute to varietal identification methods, Marker-Assisted Selection, and efficient cultivar development in upcoming programs.

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