The biomineralization of human dental enamel has resulted in a highly anisotropic and heterogeneous distribution of hydroxyapatite crystallites, which in combination with its high mineral content has resulted in one of the most durable and hardest tissues in the human body. In this study, we used position-sensitive synchrotron X-ray diffraction to quantify the spatial variation in the direction and magnitude of the preferred orientation of enamel crystallites across a whole tooth crown. Two-dimensional synchrotron X-ray diffraction images were collected with 300 μm spatial resolution over a series of six sequential tooth sections obtained from a single maxillary first premolar and were analyzed using Rietveld refinement. Both the magnitude and the direction of the crystallite orientation were found to have a high spatial heterogeneity. Areas of high crystallite alignment were directed perpendicular to the biting surfaces, which is thought to meet the functional requirements of mastication. The results may assist in our understanding of the structure–function relationship and of the evolutionary development of enamel.