Diffusion-based tractography in the optic nerve requires sampling strategies assisted by anatomical landmark information (regions of interest, ROIs). We aimed to investigate the feasibility of expert-placed, high resolution T1-weighted ROI-data transfer onto lower spatial resolution diffusion-weighted images. Slab volumes from twenty volunteers were acquired and preprocessed including distortion bias correction and artifact reduction. Constrained spherical deconvolution was used to generate a directional diffusion information grid (FOD-model (fibre orientation distribution)). Three neuroradiologists marked landmarks on both diffusion imaging variants and structural datasets. Structural ROI information (volumetric interpolated breath-hold sequence (VIBE)) was respectively registered (linear with 6/12 degrees of freedom (DOF)) onto single-shot (ss-EPI) and readout-segmented (rs-EPI) volumes respectively. All eight ROI/FOD combinations were compared in a targeted tractography task of the optic nerve pathway. Inter-rater reliability for placed ROIs among experts was highest in VIBE images (lower confidence interval 0.84 to 0.97, mean 0.91) and lower in both ss-EPI (0.61 to 0.95, mean 0.79) and rs-EPI (0.59 to 0.86, mean 0.70). Tractography success rate was highest in VIBE-drawn ROIs registered (6-DOF) onto rs-EPI FOD (70.0% over 5%-threshold, capped to failed ratio 39/16) followed by both 12-DOF registered (67.5%; 41/16) and non-registered VIBE (67.5%; 40/23). On ss-EPI FOD, VIBE ROI-datasets obtained fewer streamlines overall with each at 55.0% above 5%-threshold and with lower capped to failed ratio (6-DOF: 35/36; 12-DOF: 34/34, non-registered 33/36). The combination of VIBE-placed ROIs (highest inter-rater reliability) with 6-DOF registration onto rs-EPI targets (best streamline selection performance) is most suitable for white matter template generation required in group-studies.