Navigated brain stimulation (NBS) is a newly evolving technique. In addition to its supposed purpose, e.g., preoperative mapping of the central region, little is known about its further use in neurosurgery. We evaluated the usefulness of diffusion tensor imaging fiber tracking (DTI-FT) based on NBS compared to conventional characterization of the seed region.We examined 30 patients with tumors in or close to the corticospinal tract (CST) using NBS with the Nexstim eXimia system. NBS was performed for motor cortex mapping, and DTI-FT was performed by three different clinicians using BrainLAB iPlan® Cranial 3.0.1 at two time points. Number of fibers, tract volume, aberrant tracts, and proximity to the tumor were compared between the two methods.We recognized a higher number of fibers (1,298 ± 1,279 vs. 916 ± 986 fibers; p< 0.01), tract volume (23.0 ± 15.3 vs. 18.3 ± 14.0 cm(3); p< 0.01), and aberrant tracts (0.6 ± 0.5 vs. 0.3 ± 0.5 aberrant tracts/tracked CST; p< 0.001) when the seed region was defined conventionally, while proximity of the tracts to the tumor did not differ. While NBS-based DTI-FT is independent of the planning clinician, conventional outlining of the seed region shows generally higher variability between investigators.Conventional DTI-FT showed significant differences between the two modalities, most likely because of the more specific definition of the seed region when DTI-FT is based on NBS. Moreover, NBS-aided DTI fiber tracking is user-independent and, therefore, a method for further standardization of DTI fiber tracking.