ABSTRACTUnderstanding how plants respond to environmental stressors such as ultraviolet B (UV‐B) radiation is critical for agricultural resilience and crop improvement. Investigation of the UV‐B response in 17 cannabis accessions revealed distinct differences in morphological changes between two groups, A and B. Whole‐genome resequencing and population‐level analyses indicate that Δ9‐tetrahydrocannabinolic acid synthase (THCAS) is subject to positive selection or a selective sweep. Sequence analysis and functional characterization of THCAS revealed that two single nucleotide variations, resulting in two amino acids change at Pro333 and Ser355, which are associated with THCAS activity and potentially linked to differential plant response to UV‐B radiation. The role of THCAS in enhancing UV‐B tolerance was further demonstrated by overexpressing functional THCAS in different plant species under UV‐B stress environment. This study suggests that THCA, produced by the enzyme THCAS, acts as a shield against UV‐B radiation. Plants with higher levels of THCA exhibited greater tolerance to UV‐B exposure. Exogenous application of THCA alleviated UV‐B damage in various plant species. Our findings highlight the importance of THCAS‐mediated THCA synthesis in conferring UV‐B tolerance and provide insights into the genomic and molecular mechanisms that govern plant adaptation to UV‐B radiation.