Ozonolysis is potentially an effective method for pretreating lignocellulosic biomass to improve the production of fermentable sugars via enzymatic hydrolysis. Further understanding of the ozonolysis process and identifying specific lignin structural changes are crucial for improving the pretreatment process. Investigation into pretreatment of wheat straw using ozonolysisis is reported in this paper, with special emphasis on selective modification/degradation of lignin subunits. The ozonolysis was performed for 2 h with less than 60 mesh particles in order to achieve maximum lignin oxidation. The results showed that the lignin structure was significantly modified under these conditions, leading to higher sugar recovery of more than 50% which increased from 13.11% to 63.17% corresponding to the control and ozone treated samples, respectively. Moisture content was found to be an important parameter for improving sugar recovery. Ninety percent (w/w) moisture produced the highest sugar recovery. The concentration of acid soluble lignin in the ozone treated sample increased from 4% to 11% after 2 h treatment. NMR analysis revealed that the S2/6 and G2 lignin units in the wheat straw were most prone to oxidation by ozone as the concentration of aromatic units decreased while the carboxylic acids became more abundant. The experimental data suggest the degradation of β-O-4 moieties and aromatic ring opening in lignin subunits. The pyrolysis-gas chromatography/mass spectrometry results revealed that the rate of lignin unit degradation was in the following order: syringyl > guaiacyl > p-hydroxyphenyl. Long ozone exposure resulted in few condensed lignin structure formation. In addition, the formation of condensed units during this process increased the activation energy from ASTM-E, 259.74 kJ/mol; Friedman-E, 270.08 kJ/mol to ASTM-E, 509.29 kJ/mol; Friedman-E, 462.17 kJ/mol. The results provide new information in overcoming lignin barrier for lignocellulose utilization.