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The pretreatment of straw directly influences the following conversion efficiency. Hence, comprehensively understanding the changes in the pretreatment process is of great significance. This study used microcomputed tomography as a characterization technique to study the changes in the microstructure of dilute-acid-pretreated corn stalks. The optimal accelerating voltage for scanning and the optimal parameters for image reconstruction were determined. Rapid and noninvasive three-dimensional visual characterization of the corn stalk internal structure could be performed using microcomputed tomography, which has several advantages over conventional methods. After processing the obtained images, the microstructural changes of the stalk, including those in the thickness distribution, porosity, separation distribution, and pore connectivity as well as the X-ray mean attenuation coefficient were calculated and compared with those reported in the literature. Hence, microcomputed tomography is a feasible and nondestructive characterization method for better understanding of the effects of acid pretreatments on corn stalks and for optimizing pretreatment parameters.