Integration of the neural processes in the human brain is realized through interconnections that exist between different neural centers. These interconnections take place through white matter pathways. White matter tractography is currently the only available technique for the reconstruction of the anatomical connectivity in the human brain noninvasively and in vivo. The trajectory and terminations of white matter pathways are estimated from local orientations of nerve bundles. These orientations are obtained using measurements of water diffusion in the brain. In this article, the techniques for estimating fiber directions from diffusion measurements in the human brain are reviewed. Methods of white matter tractography are described, together with the current limitations of the technique, including sensitivity to image noise and partial voluming. The applications of white matter tractography to the topographical characterization of the white matter connections and the segmentation of specific white matter pathways, and corresponding functional units of gray matter, are discussed. In this context, the potential impact of white matter tractography in mapping the functional systems and subsystems in the human brain, and their interrelations, is described. Finally, the applications of white matter tractography to the study of brain disorders, including fiber tract localization in brains affected by tumors and the identification of impaired connectivity routes in neurologic and neuropsychiatric diseases, are discussed. Copyright © 2010 John Wiley & Sons, Ltd.