The changes occurring during the depolymerization of lignocellulosic biomasses are not yet fully understood. Synchrotron micro-Fourier transform infrared (mu-FTIR), Raman spectroscopy (RS), X-ray diffraction (XRD), and X-ray fluorescence (XRF) were applied for better characterization of wheat straw fibers during a continuous pretreatment process in terms of conditioning (C), extrusion (E), steam explosion (SE), and enzymatic hydrolysis (EH). mu-FTIR revealed functional groups as phenylpropanoid polymers, ethers, and aliphatic alcohol. RS detected acetoacetate, methyl and phenol groups after SE. The crystallinity index and silica content were also determined.