Given that the glycerol organosolv pretreatment (GOP) can effectively improve the hydrolyzability of various lignocellulosic substrates, physicochemical changes of the substrate before and after the pretreatment was characterized to elucidate what is responsible for it. The effect of GOP on the main components and hydrolyzability of wheat straw was revisited. Results demonstrate that the GOP should be a promising candidate for the current pretreatment. Then the composition and structure of substrates was measured at multi-dimensional scales by using various analytic equipment such as TGA, SEM, AFM, CLSM, FTIR, XRD and solid-state CP/MAS C-13 NMR. This paper reports some new insights on the mechanism behind that, which can be beneficial for further development, optimization, and scale-up of the GOP process. (C) 2015 Elsevier Ltd. All rights reserved.