Effects of the pretreatment of wheat straw by steam explosion and ethanol were evaluated relative to the structural changes of lignin from the pretreated pulp. The lignin from steam explosion pulp (LS), lignin from steam blasting residual liquid (LL), lignin from ethanol pretreatment pulp (LE), lignin from black liquor (LB), and lignin from wheat straw (LW) were separated, and the structural characteristics of the lignin fractions were compared based on analyses of Fourier transform-infrared, ultraviolet, thermogravimetric, and 1H and 13C nuclear magnetic resonance spectra. The proportions of the three structural units in all lignin fractions clearly changed during the pretreatment process because of inter-conversion reactions. The conjugated structure of lignin was destroyed in the pretreatment process and was also affected by the alkali extraction process. The alcoholic hydroxyl links on the aliphatic side chain were partly transformed into carbonyl groups during ethanol pretreatment. Demethoxylation occurred in all lignin fractions during the ethanol pretreatment and steam explosion process. The thermal stability of the LB fraction was relatively high because of the condensation reaction.