Crack initiation in AISI 316 stainless steel has been investigated. Persistent Slip bands (PSBs) were characterized using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). PSBs on the surface of the material increase the surface roughness and result in crack initiation. EBSD data from near the crack initiation region were used to correlate the global and local misorientations of the grains, plastic deformation, and Schmid Factor with the fatigue life of specimens. The crack initiation region was found to have the highest misorientations. The region near crack initiation was found to have more plastic deformation, which was severe in specimens loaded with higher stresses. The Kernel Average Misorientation (KAM) and Grain Reference Orientation Deviation (GROD) maps from the EBSD data were investigated for specimens that failed at different fatigue cycles. It was found that the interaction of high dislocation density, substructuring and misorientation of low-angle grain boundaries in the region of plastic deformation resulted in the fatigue crack initiation.