BACKGROUND AND PURPOSE: There is limited standardization of acquisition and processing methods in diffusion tractography for pre-surgical planning, leading to a range of approaches. In this study, a number of representative acquisition variants and post processing methods are considered, to assess their importance when implementing a clinical tractography program. METHODS: Diffusion MRI was undertaken in ten healthy volunteers, using protocols typical of clinical and research scanning: a 32-direction diffusion acquisition with and without peripheral gating, and a non-gated 64 diffusion direction acquisition. All datasets were post-processed using diffusion tensor reconstruction with streamline tractography, and with constrained spherical deconvolution (CSD) with both streamline and probabilistic tractography, to delineate the cortico-spinal tract (CST) and optic radiation (OR). The accuracy of tractography results was assessed against a histological atlas using a novel probabilistic Dice overlap technique, together with direct comparison to tract volumes and distance of Meyer's loop to temporal pole (ML-TP) from dissections studies. Three clinical case studies of patients with space occupying lesions were also investigated. RESULTS: Tracts produced by CSD with probabilistic tractography provided the greatest overlap with the histological atlas (overlap scores of 44% and 52% for the CST and OR, respectively) and best matched tract volume and ML-TP distance from dissection studies. The acquisition protocols investigated had limited impact on the accuracy of the tractography. In all patients, the CSD based probabilistic tractography created tracts with greatest anatomical plausibility, although in one case anatomically plausible pathways could not be reconstructed without reducing the probabilistic threshold, leading to an increase in false positive tracts. CONCLUSIONS: Advanced post processing techniques such as CSD with probabilistic tractography are vital for pre-surgical planning. However, overall accuracy relative to dissection studies remains limited.