MRI-based white matter tract estimation using probabilistic tractography is valuable for presurgical planning. In this work, we investigate three factors influencing the reproducibility of probabilistic tractography: the tract threshold used to visualise the analysis results in the final tractography image, the manually drawn seed region, and the end region of the tracts. A compromise between low risk and high reproducibility has to be found for the tract threshold. The end region definition was found to be the most important parameter for the inter-user reproducibility of restricted tracts. Good inter-user reproducibility for the unrestricted tracts is achievable for similar seed regions.