Diffusion tensor imaging (DTI) is an MRI-based technique that delineates white matter tracts in the brain by tracking the diffusion of water in neural tissue. This methodology, known as “tractography”, has been extensively applied in clinical neuroscience to explore nervous system architecture and diseases. More recently, tractography has been used to assist with neurosurgical targeting in functional neurosurgery. This review provides an overview of DTI principles, and discusses current applications of tractography for improving and helping develop novel deep brain stimulation (DBS) targets.