The evolution of microstructures of X20CrMoV12.1 heat-resistant steels exposed at 545 ℃ after long time running was investigated through scanning electron microscope and transmission electron microscope in this study. Notch fatigue strength and hardness of the materials running for different time were also measured. The results show that the microstructures became degraded significantly after long time running. The microstructures were changed from the original martensite lamellar/fine carbides to subgrains/coarsened spherical carbides along boundaries. With increasing running time at high temperature, the microhardness and the notch fatigue strength gradually decreased. The reduction of Cr, Mo and V in matrix results in the loss of solidification strengthening. The coarsened spherical carbides along boundaries reduce grain boundary strength of the material. As a result, the reliability of the X20CrMoV12.1 steels under a long-term service condition at high temperature is degraded.