Lignin preparation with high yield (85.71%) and purity (96.97%) from wheat straw was obtained by ball milling treatment and direct acidic dioxane extraction. This procedure gives a material that is representative of the whole lignin of wheat straw. The lignin fraction is distinguished by high β-O-4' structures and small amounts of condensed units (β-5', 5-5', and β-1') enriched in guaiacyl units. p-Coumaric acid is linked to lignin via γ-ester bond and a few ether bonds such as β-O-4', whereas ferulic acid is linked to lignin by ether bonds, and its dehydrodimers (5-5'/8-O-4') are also incorporated into lignin polymer.