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Chemical characterization of kraft lignin (KL) from mixed hardwoods (Acacia spp. from Vietnam and mixed hardwoods (mainly Quercus spp.) from Korea) was conducted for its future applications. To compare the structural changes that occurred in KL, two milled wood lignins (MWLs) were prepared from the same hardwood samples used in the production of KL. Elemental analysis showed that the MWL from acacia (MWL-aca) and mixed hardwood (MWL-mhw) had almost similar carbon content, methoxyl content, and C9 formula. KL had high carbon content but low oxygen and methoxyl contents compared to MWLs. The C9 formula of KL was determined to be C9H7.29O2.26N0.07S0.12(OCH3)1.24. The Mw of KL and MWLs was about 3000 Da and 12,000–13,000 Da, respectively. The structural features of KL and MWLs were investigated by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectrometry (1H, 13C NMR). The analyses indicated that KL underwent severe structural modifications, such as γ-carbon cleavage, demethylation, and polycondensation reactions during kraft pulping, which resulted in increased aromatic content and decreased aliphatic content. The main linkages in lignin, β-O-4 moieties, were hardly detected in the analysis as these linkages were extensively cleaved by nucleophilic attack of SH- and OH- during pulping. [ABSTRACT FROM AUTHOR]