BACKGROUND AND PURPOSE: Diffusion tensor imaging (DTI) tractography reconstruction of white matter pathways can help guide brain tumor resection. However, DTI tracts are complex mathematical objects and the validity of tractography-derived information in clinical settings has yet to be fully established. To address this issue, we initiated the DTI Challenge, an international working group of clinicians and scientists whose goal was to provide standardized evaluation of tractography methods for neurosurgery. The purpose of this empirical study was to evaluate different tractography techniques in the first DTI Challenge workshop. METHODS: Eight international teams from leading institutions reconstructed the pyramidal tract in four neurosurgical cases presenting with a glioma near the motor cortex. Tractography methods included deterministic, probabilistic, filtered, and global approaches. Standardized evaluation of the tracts consisted in the qualitative review of the pyramidal pathways by a panel of neurosurgeons and DTI experts and the quantitative evaluation of the degree of agreement among methods. RESULTS: The evaluation of tractography reconstructions showed a great interalgorithm variability. Although most methods found projections of the pyramidal tract from the medial portion of the motor strip, only a few algorithms could trace the lateral projections from the hand, face, and tongue area. In addition, the structure of disagreement among methods was similar across hemispheres despite the anatomical distortions caused by pathological tissues. CONCLUSIONS: The DTI Challenge provides a benchmark for the standardized evaluation of tractography methods on neurosurgical data. This study suggests that there are still limitations to the clinical use of tractography for neurosurgical decision making.