To investigate the effect of temperature on microcracking mechanisms of modified 9Cr-1Mo steel, low cycle fatigue tests were performed at 350 C and room temperature (RT). At RT, the cyclic stress response at low strain amplitude (<0.5%) is controlled by friction stress while dominated by back stress at high strain amplitude. By comparison, the disappearance of sub-grain boundaries induced by dislocation annihilation occurs at 350 C, which contributes to the decrease in back stress, resulting in more significant effects of back stress compared to RT. As a result of the accumulated fatigue damage, the distance between two parallel extrusions at 350 C is larger than that at RT, which is ascribed to the disappearance of low angle sub-grain boundaries due to dislocation annihilation at 350 C. Due to the disappearance of small-angle grain boundaries at 350 C, microcracks initiate along high-angle grain boundaries at 350 C instead of along low-angle grain boundaries at RT.