From Wild-Growing Plants to Promising Candidates for Hemp Variety Development: Morphological and Phytochemical Characterization of Macedonian Cannabis sativa L.

Background: Wild-growing Cannabis sativa L. represents an underexplored source of hemp-type plant material, but its value for future selection depends on standardized morphological and phytochemical characterization. This study evaluated cultivated Macedonian Cannabis sativa plants of wild-origin (CsW) in comparison with selected commercial cultivars and literature-reported European hemp varieties, based on the assessment of phenotypic differentiation and cannabinoid diversity. Methods: Seeds from five wild-growing plant groups from two N. Macedonian regions, Kočani (W1, W2, W6) and Strumica (W3 and W4), were planted outdoors together with four commercial cultivars with differing cannabinoid composition. Morphological traits were assessed using selected UPOV descriptors and multivariate analysis. Cannabinoids were determined by HPLC-DAD. Results: UPOV-based PCA clearly differentiated CsW from the commercial cultivars (R2X = 0.92, Q2 = 0.86), while the expanded comparative UPOV analysis indicated phenotypic affinity with registered hemp-type material, particularly Kompolti, supporting its potential relevance as a phenotypic reference in future DUS-oriented evaluation. Among the cultivated wild-origin groups, W1 from the Kočani region showed the highest proportion of female plants (10:1), representing a favorable agronomic characteristic for flower-oriented production. All CsW were CBD-predominant chemotype III class, with total CBD of 5.14–7.61%, while total Δ9-THC did not exceed 0.20%. This CBD/THC balance positioned the CsW within the higher-CBD range reported for selected European hemp cultivars, while maintaining a favorable low-THC profile. Conclusions: CsW represent morphologically distinct, CBD-rich and low-THC plant material displaying characteristics consistent with a hemp-type profile. The combined UPOV and cannabinoid data support its relevance as a promising resource for further agronomic evaluation, phytochemical selection and future registration-oriented hemp research.