Biological valorization of biomass most often depends on the efficient reduction of plant cell wall recalcitrance and conversion of lignin - the most recalcitrant constituent - to fuels, chemicals and/or value-added substances. Lignin conversion to fuels and value-added chemicals requires a sound understanding of the structure of lignin before and after different pretreatments. In the current work, an effort has been made to compare the structural differences in isolated pine lignin after one- (alkaline) and two-step (alkaline/dilute acid and alkaline/steam explosion) pretreatments. Our results indicate removal of the low molecular weight fraction of lignin after an initial alkaline pretreatment. A subsequent dilute acid pretreatment resulted in the loss of lignin inter-unit linkages such as beta-O-4' aryl ethers. However, with a steam explosion pretreatment, lignin exhibited a competing condensation process leading to increased condensed lignin structures.