Self-assembled colloidal crystals have attracted major attention because of their potential as low-cost three-dimensional (3D) photonic crystals. Although a high degree of perfection is crucial for the properties of these materials, little is known about their exact structure and internal defects. In this study, we use tomographic scanning transmission X-ray microscopy (STXM) to access the internal structure of self-assembled colloidal photonic crystals with high spatial resolution in three dimensions for the first time. The positions of individual particles of 236 nm in diameter are identified in three dimensions, and the local crystal structure is revealed. Through image analysis, structural defects, such as vacancies and stacking faults, are identified. Tomographic STXM is shown to be an attractive and complementary imaging tool for photonic materials and other strongly absorbing or scattering materials that cannot be characterized by either transmission or scanning electron microscopy or optical nanoscopy.