A significant challenge in biofuel production is the inefficient hydrolysis of lignocellulosic materials into simple sugar for conversion into fuel ethanol. This low efficiency results in part from lignin restricting the access of degradative enzymes to cellulose. Wheat straws exposed to electron beams ranging from 0 to 100 kGy were examined to determine effect of divided irradiation (25 kGy in 4 tandem doses) vs. single irradiation (100 kGy). Yields of reducing sugars such as glucose and xylose after divided irradiation (51.1%) showed only a small increase relative to the control (40.9%), whereas in single irradiation, the yields of reducing sugars substantially increased to 74.9%. Results of X-ray diffraction showed crystallinity of cellulose slightly decreased from 43.0 to 38.8% after divided irradiation. Significant decrease to 34.1% was observed after single irradiation. Removal of hemicelluloses and modification of lignin polymer were also observed. These results indicate electron beam irradiation has destructive influence on both physical and chemical properties of wheat straw, thereby increasing accessibility of the cellulose surface to enzymatic hydrolysis and theoretically enable more efficient cellulose hydrolysis. Moreover, divided irradiation did not show a significant influence on enzymatic hydrolysis; hence, the single irradiation process is more effective than divided irradiation for increasing hydrolysis efficiency.