The brain contains more than 100 billion neurons that communicate with each other via axons for the formation of complex neural networks. The structural mapping of such networks during health and disease states is essential for understanding brain function. However, our understanding of brain structural connectivity is surprisingly limited, due in part to the lack of noninvasive methodologies to study axonal anatomy. Diffusion tensor imaging (DTI) is a recently developed MRI technique that can measure macroscopic axonal organization in nervous system tissues. In this article, the principles of DTI methodologies are explained, and several applications introduced, including visualization of axonal tracts in myelin and axonal injuries as well as human brain and mouse embryonic development. The strengths and limitations of DTI and key areas for future research and development are also discussed.