The profile samples prepared by focused ion beam (FIB) in crack initiation region (CIR) and fish-eye (FiE) region of failed specimens subjected to rotary bending (RB) and ultrasonic axial (UL) fatigue testing with various stress ratios (R) were observed by transmission electron microscopy (TEM) with selected area electron diffraction (SAD) detection for two high-strength steels. The grain size and the thickness of nanograin layer along the crack growth path in CIR underneath fine-granular-area (FGA) were measured for the cases of R 0 and the FiE region outside CIR for either negative or positive stress ratio cases, which suggests that the formation of nanograin layer in the FGA region is due to the numerous cyclic pressing (NCP) process and the plastic deformation ahead of the crack tip may cause certain extent of microstructure deformation but is insufficient to form nanograin layer on crack surfaces.