The hierarchical structure of bone makes the X-ray microdiffraction scanning techniques one of the most effective tool to investigate the structural features of this tissue at different length scales: the atomic/nanometer scale of the X-ray scattering signals and the macroscopic scale of the scanned sample area. The potentiality of the microdiffraction approach has been verified also by investigations on tissue-engineered bone substitutes used to repair large hard bone defects. The aim of this review is to present the most representative and recent results obtained through high-resolution scanning microdiffraction techniques studying both natural and tissue-engineered bone. The rapid evolution of the instrumental set-ups and the advanced methods of data analysis are described. Recent examples in which X-ray microbeams were used for imaging quantitative features of natural bone tissue and engineered bone substitutes are presented along with the qualitative and quantitative information extracted from the two-dimensional patterns collected on bone samples and on ex vivo cell seeded bioceramic implants. Thanks to the microdiffraction approach, several aspects of the mechanisms leading to the generation of the new bone, coupled to the scaffold resorption in the tissue-engineered constructs, have been tentatively interpreted. The potential of X-ray microdiffraction as an imaging tool in the field of bone tissue engineering is discussed and the key role of high-spatial resolution, availability of automatic tools (for dealing with the huge amount of experimental data) and advanced analysis techniques is elucidated. Finally, future perspectives in the field are presented.