Cotton fibers have complex structures despite being composed almost exclusively of the molecule cellulose. Cellulose molecules, composed of d-glucose residues, are arranged in nano-sized crystals, or crystallites. The fundamental unit of the crystal is the unit cell that is repeated along its edges, along with the atoms inside, to create the entire crystal. Crystallites are grouped into microfibrils that, in turn, are clustered into sheet-like structures that wrap around the lumen or vacuole at various angles. These layers of primary wall sheets are themselves surrounded by a winding layer, the primary wall, and a cuticle. This chapter focuses on X-ray diffraction to interpret cotton fiber structure. Patterns are shown from electron diffraction, laboratory powder X-ray diffraction, and synchrotron X-ray diffraction for both single and bundled fibers. Single-fiber and fiber bundle patterns differ because of the varied angle of the microfibrils to the main fiber axis. The bundle patterns include many of the varied angles, but single fibers have just one major angle when a micron-sized X-ray beam is used. Also shown are computed diffraction patterns based on the known atom positions. Widely used methods for X-ray analysis are flawed. Instead, interpretations of the patterns can follow the logic of “what modifications are needed to the computed ideal pattern” so that it best resembles the experimental pattern. The Rietveld powder diffraction method refines variables such as crystallite size, unit cell dimensions, and amount of amorphous material. However, there are many other possible variables in Rietveld software, more than cotton data can support.