Quenched and tempered Cr-Mo steel is commonly used in the construction of seamless hydrogen storage vessels. The resistance of such material to hydrogen embrittlement is significantly influenced by its strength and microstructure, both of which are closely related to the tempering temperature during heat treatment. Thus, optimizing the tempering temperature can potentially enhance the performance of Cr-Mo steel in high-pressure gaseous hydrogen and extend the fatigue life of seamless hydrogen storage vessels. In this study, seamless hydrogen storage vessels designed for a pressure of 50 MPa were tempered at temperatures ranging from 580 °C to 660 °C. Specimens sampled from these vessels were subjected to fatigue crack growth rate (FCGR) tests and threshold stress intensity factor for hydrogen-assisted cracking (KIH) tests in 50 MPa gaseous hydrogen, and hydrogen permeation tests were also conducted. Additionally, a fatigue life analysis of the vessels was performed using the fracture mechanics method based on the test results of Cr-Mo steel. The results indicate that increasing tempering temperature reduces the FCGR and improves KIH of Cr-Mo steel in high-pressure gaseous hydrogen, thereby extending the fatigue life of seamless hydrogen storage vessels. Simultaneously, the increase in tempering temperature leads to a greater quantity and more dispersed distribution of precipitated carbides. These carbides, serving as irreversible hydrogen traps, effectively hinder the diffusion of hydrogen atoms, which is a key factor contributing to the enhanced resistance of Cr-Mo steel to hydrogen embrittlement with higher tempering temperatures.