Industrial hemp （Cannabis sativa L.） is a high-value cash crop with applications in papermaking， healthcare， textiles， and other industries. The development of new industrial hemp germplasm is crucial for both breeding programs and functional genomic research. In this study， we constructed a mutant library of the variety ZHONGDAMAZI 4 （ZD4） using ethyl methanesulfonate （EMS） mutagenesis. By establishing a gradient of eight EMS concentrations， we determined that a 0.8% EMS concentration was optimal for inducing mutations in ZD4. After treating 2000 seeds with EMS solution， a total of 644 M₁ seedlings were obtained， with a germination rate of 32.2%. Ninety individual plants exhibited variations in leaf color， inflorescence， and plant height， resulting in a mutation rate of 13.98%. High-performance liquid chromatography （HPLC） was used to measure the cannabidiol （CBD） content of the M₁ plants， resulting in the identification of 3 high-CBD and 2 low-CBD mutants. Sequencing analysis didn’t reveal sequence variations in the coding and promoter sequences 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 enzymes varied significantly compared to the control. It is inferred that EMS treatment induced mutations in regulatory genes affecting the CBD metabolic pathway in the mutants， thereby regulating the transcriptional expression and resulting in significant changes in CBD content. This study provides germplasm resources for industrial hemp breeding through the establishment of an EMS mutant library and lays a solid foundation for further elucidating the regulatory mechanisms of cannabinoid biosynthesis and related genes.