Functional stereotactic neurosurgery by means of deep brain stimulation or ablation provides an effective treatment for movement disorders, but the outcome of surgical interventions depends on the accuracy by which the target structures are reached. The purpose of this pilot study was to evaluate the feasibility of diffusion tensor imaging (DTI) based probabilistic tractography of deep brain structures that are commonly used for pre- and perioperative targeting for functional neurosurgery. Three targets were reconstructed based on their significance as intervention sites or as a no-go area to avoid adverse side effects: the connections propagating from the thalamus to (1) primary and supplementary motor areas, (2) to somatosensory areas and the cerebello-thalamic tract. We evaluated the overlap of the reconstructed connectivity based targets with corresponding atlas based data, and tested the inter-subject and inter-scanner variability by acquiring repeated DTI from four volunteers, and on three MRI scanners with similar sequence parameters.Compared to a 3D histological atlas of the human thalamus, moderate overlaps of 35-50% were measured between connectivity- and atlas based volumes, while the minimal distance between the centerpoints of atlas and connectivity targets was 2.5 mm. The variability caused by the MRI scanner was similar to the inter-subject variability, except for connections with the postcentral gyrus where it was higher. While cerebello-thalamic tractography resolved the anatomically correct trajectory of the tract individually, high volumetric variability was found across subjects and between scanners. DTI can be applied in the clinical, preoperative setting to reconstruct the cerebello-thalamic tract and to localize subdivisions within the lateral thalamus. In our pilot study, such subdivisions moderately matched the borders of the ventrolateral-posteroventral (VLpv) nucleus and the ventral-posterolateral (VPL) nucleus. Limitations of the currently used standard DTI protocols were exacerbated by large scanner-to-scanner variability of the connectivity-based targets.