To reduce the recalcitrance and enhance enzymatic activity, dilute H2SO4 pretreatment was carried out on Alamo switchgrass (Panicum virgatum). Ball-milled lignin was isolated from switchgrass before and after pretreatment. Its structure was characterized by C-13, HSQC, and P-31 NMR spectroscopy. It was confirmed that ball-milled switchgrass lignin is of HGS type with a considerable amount of p-coumarate and felurate esters of lignin. The major ball-milled lignin interunit was the beta-O-4 linkage, and a minor amount of phenylcoumarin, resinol, and spirodienone units were also present. As a result of the acid pretreatment, there was 36% decrease of beta-O-4 linkage observed. In addition to these changes, the S/G ratio decreases from 0.80 to 0.53.