AbstractHemp (Cannabis sativa L.) shiv has great potential for the production of bio-composites as a reinforcement material. To gain more information about hemp shiv, this research studied the influence of gender on the physical and mechanical properties of the fiber cell wall in the shiv of three dioecious hemp plant varieties by optical microscopy, image analysis software, WXRD, and nanoindentation. The results show that a hemp plant’s gender greatly influences the properties of hemp shiv. While long and thin in female hemp shiv, the fibers are shorter with a larger diameter in male hemp shiv. In addition, the cell walls in female shiv are thinner than those in male shiv. The microfibril angle (MFA), relative degree of crystallinity, elastic modulus, and hardness values of fiber cell walls as well as the lignin content in male hemp plants are higher than those in female hemp plants. Besides, the relationship between mechanical properties and MFA do not align with those observed in previous research, which shows that the gender of an individual plant has a greater effect on the mechanical properties of the fiber cell wall than does its MFA. Thus, when the fiber from this dioecious plant is investigated or used, the sex of the plant should be known and considered.