The high processing cost of lignocellulosic ethanol is one of the most important barriers to its profitable commercialization. Pretreatments have been used to change the structure of biomass significantly and to improve sugar and ethanol yield. Great efforts have been made to understand the structural changes of biomass during these processes, including the molecular assembly of crystalline cellulose. Wide-angle and small-angle X-ray scattering are powerful techniques in studying the biomass structure at a molecular level. In this review, after we introduce the basic structure of lignocellulosic biomass, the effects of commonly used pretreatment methods on biomass structure, and the principle of X-ray scattering technique, the application of X-ray scattering, including studies of crystallinity, crystallite size, orientation distribution, and pore structure, and the related results in biomass conversion are summarized and discussed. Future study of biomass with X-ray scattering also is proposed.