Agricultural residues and energy crops are considered potential feedstocks for bioethanol production because of their high availability and energy potential as well as relatively low cost. Previous studies have shown that pelleting biomass feedstocks could increase their bulk density, thus increasing ease of handling and decreasing cost of handling and transportation. The pelleting process has also been shown to have a positive impact on the sugar yield of biomass. However, the effects of the pelleting process on biomass structure have not yet been studied. Therefore, the objective of this study was to investigate the impact of dilute acid pretreatment and the pelleting process on biomass structure of cellulosic materials, including crystallinity index (CrI,%) measured by the x-ray diffraction (XRD) method, structure of constituents and chemical changes determined by Fourier transform infrared spectroscopy (FTIR) and solid-state cross-polarization/magic angle spinning (CP/MAS) 13C NMR spectroscopy, morphological structure determined by scanning electron microscopy (SEM), and thermal properties determined by thermogravimetric analysis (TGA). Wheat straw, big bluestem, corn stover, and photoperiod-sensitive sorghum were used for this study. Pelleting did not have a significant effect on the pattern of FTIR spectra and solid-state 13C NMR spectra of biomass. XRD analysis showed that biomass crystallinity increased after dilute acid pretreatment and the pelleting process. Based on SEM analysis of biomass, dilute acid pretreatment and pelleting enhanced the removal of the softened surface region of biomass. TGA analysis showed that the decomposition temperature of pelleted biomass was slightly higher than that of corresponding unpelleted biomass, indicating that the pelleted biomass was more thermally stable than the unpelleted biomass.