Purpose Successful utilization of anatomical templates in the evaluation of diffusion-weighted neuroimaging-studies requires accurate registration of intra-individual datasets. We investigated the feasibility of structural MRI image registration onto single-shot and read-out segmented echo-planar diffusion-weighted imaging variants for use in tractographic samplings of the visual system, in particular the optic nerve. Methods Slab volumes of the optic nerve pathway from thirteen volunteers were acquired and preprocessed. Three neuroradiologists marked landmarks (ROIs, regions of interest) on two diffusion-weighted and one structural dataset. Structural ROIs were respectively registered (6/12 degrees of freedom, DOF) onto single-shot (ss-EPI) and readout-segmented (rs-EPI) volumes. All six ROI/FOD (fibre orientation distribution) combinations underwent a targeted tractography task (MRtrix3: iFOD2). Results Inter-rater reliability for ROI-placement was highest in VIBE images (0.66 to 0.95, mean 0.85) and lower in both ss-EPI (mean 0.77) and rs-EPI (0.46 to 0.84, mean 0.64). Sufficient FOD generation in the optic nerve was successful in 12/26 of all cases for ss-EPI volumes (46.2% of cases) and in 18/26 for rs-EPI volumes (69.2%). Spatial shift of VIBE-drawn ROI-coordinates after 6-DOF registration was highest for ss-EPI-targets (medians: 1.15 to 1.4 mm; rs-EPI 0.82 to 0.93 mm), whereas 12-DOF registration caused less spatial shifts (ss-EPI: 0.64 to 1.03 mm; rs-EPI: 0.58 mm to 0.79 mm). Tractography results revealed no significant differences between ss-EPI and rs-EPI sequences on cases with mutual generation of sufficient FODs (n=10). Conclusion Structurally-placed ROIs (highest inter-rater reliability) with 6-DOF registration onto rs-EPI targets (highest FOD-generation rate) seems most suitable for white matter template generation.