Internal hydrogen can influence the fatigue life, crack growth rate, and crack morphology of austenitic stainless steel, but little is known about the effect of internal hydrogen on fatigue crack initiation sites. To determine the effect of internal hydrogen on the microstructural fatigue crack initiation sites, the location of small fatigue cracks was evaluated with respect to the microstructural features in notched middle tension M(T) 316L specimens both with and without pre-charged hydrogen. The notches of the M(T) specimens were electropolished prior to fatigue testing to facilitate post-test analysis. Fatigue tests were performed with the same constant load amplitude and an R-ratio of 0.1 for specimens with and without internal hydrogen. The fatigue tests were interrupted after a minimal amount of cracking was detected using the direct current potential difference (DCPD) technique. The microstructural locations of the small fatigue cracks were then evaluated with scanning electron microscopy imaging and electron backscatter diffraction (EBSD). Several small transgranular fatigue cracks initiated in the notches of specimens both with and without internal hydrogen. These transgranular cracks always intersected grain boundaries, twin boundaries, and/or triple points indicating that these microstructural features are the critical locations for crack initiation. The transgranular cracks did not propagate along the prominent slip traces. There was no discernible effect of hydrogen on the microstructural sites of fatigue crack initiation in 316L.