Industrial hemp (Cannabis sativa L.) is a high-value economic crop used in papermaking, healthcare, textiles, and other fields. Developing new germplasm of industrial hemp is essential for breeding, innovative germplasm resources, and functional genomic research. In this study, a mutant library of ZHONGDAMAZI 4 (ZD4) was constructed using ethyl methanesulfonate (EMS). By setting up gradients of eight concentrations of EMS, it was determined that the 0.8% EMS treatment concentration had the best mutagenic effect on ZD4. After mutagenizing 2000 seeds of ZD4, a total of 644 M1 seedlings emerged, with an emergence rate of 32.2%. Among them, 90 individual plants exhibited variations in leaf color, inflorescence, and plant height, with a mutation rate of 13.98%. High-performance liquid chromatography (HPLC) was used to determine the cannabidiol (CBD) content of the M1 plants, resulting in the identification of 3 high-CBD and 2 low-CBD mutants. Sequencing revealed that the DNA sequences as well as the promoter regions of five key cannabinoid biosynthetic enzyme genes (CsPT4, CsOAC1, CsOLS1, CsCBDAS, and CsTHCA) in the CBD content mutants remained unchanged. However, the expression levels of these five enzymes showed significant changes compared to the control. It is inferred that EMS treatment led to mutations in regulatory genes affecting the CBD metabolic pathway in mutants, thereby regulating the expression of enzymes, ultimately resulting in significant changes in CBD content. This study provides excellent germplasm resources for industrial hemp breeding through the construction of an EMS mutant library and lays a solid foundation for further elucidating the regulatory mechanisms of cannabinoid biosynthesis and related genes.