AbstractAspen wood was subjected to autohydrolysis as a pre-treatment to characterize the structural changes occurring in lignin fractions during the pre-treatment process. Milled wood lignin (MWL) was isolated from both the native aspen wood and hydrolyzed wood chips, and its structural features were characterized by Fourier transform infrared (FT-IR), quantitative 13C, two-dimensional heteronuclear single quantum coherence (2D HSQC), and 31P nuclear magnetic resonance (NMR) spectroscopies, gel permeation chromatography/multi-angle laser light scattering (GPC-MALLS), and thermal analysis. The lignin remaining in the hydrolyzed wood chips revealed more phenolic OH groups, fewer aliphatic OH groups, higher syringyl/guaiacyl ratios (S/G), higher molecular weights, and narrower polydispersities than the native lignin of aspen wood. The inter-unit linkages of β-O-4 were noticeable cleaved, but the condensed structures in the lignin formed when undergoing autohydrolysis of high severity, resulting in elevated amounts of C-C linkages. Moreover, it was found that autohydrolysis promoted the removal of -OCH3 groups and increased the thermal stability of lignin fractions.