AbstractLow-pressure steam explosion (LPSE) combined with alkaline peroxide (AP) pretreatment was first employed to separate hemicellulose from Lespedeza stalks. The monosaccharide composition and molecular weight distribution of the obtained hemicellulose fractions were characterized in this study. The results show that the hemicellulose extracted from Lespedeza stalks consisted of xylose, glucose, galactose, and mannose, which was a mixture of arabinoxylans and xyloglucans or β-glucans. The yield of hemicellulose fractions after AP pretreatment ranged from 11.2% (2.5% hydrogen peroxide (H2O2), w/v for 12 h) to 12.2% (3.3% H2O2, w/v for 72 h). The molecular weight of hemicellulose decreased from 2,458 g/mol to 1,984 g/mol after AP pretreatment, indicating its degradation reaction. The structure of hemicellulose was analyzed by Fourier transform infrared spectroscopy, proton nuclear magnetic resonance, and heteronuclear single quantum coherence. The AP pretreatment partially cleaved the backbone and the ether linkage between lignin and hemicelluloses. Also, branched-chain α-D-arabinofuranosyl in which β-D-xylose substituted at the C-3 position (monosubstituted) was removed, illustrating a partial debranching reaction. Therefore, the combination of low-pressure steam explosion and alkaline peroxide pretreatment (LPSE-AP) is an effective pretreatment method to separate hemicellulose from Lespedeza stalk.