Abstract Tractography is the standard tool for automatic delineation of white matter tracts from diffusion weighted images. However, the output of tractography often requires post-processing to remove false positives and ensure a robust delineation of the studied tract, and this demands expert prior knowledge. Here we demonstrate how such prior knowledge, or indeed any prior spatial information, can be automatically incorporated into a shortest-path tractography approach to produce more robust results. We describe how such a prior can be automatically generated (learned) from a population, and we demonstrate that our framework also retains support for conventional interactive constraints such as waypoint regions. We apply our approach to the open access, high quality Human Connectome Project data, as well as a dataset acquired on a typical clinical scanner. Our results show that the use of a learned prior substantially increases the overlap of tractography output with a reference atlas on both populations, and this is confirmed by visual inspection. Furthermore, we demonstrate how a prior learned on the high quality dataset significantly increases the overlap with the reference for the more typical yet lower quality data acquired on a clinical scanner. We hope that such automatic incorporation of prior knowledge and the obviation of expert interactive tract delineation on every subject, will improve the feasibility of large clinical tractography studies.