Acid-catalysed crude glycerol (ACG) pretreatment was carried out at 110 °C and 130 °C for mild fractionation of sugarcane bagasse into fermentable sugars and high-quality lignin. ACG pretreatment at 110 °C led to sugar yields of 71%-74%, comparable to those with acid-catalysed reagent-grade glycerol (AG). ACG pretreatment removed more lignin (53%-75%) than AG pretreatment (38%-49%), likely due to the presence of organic impurities in ACG. Hence, 28% more lignin was recovered from ACG pretreatment hydrolysate than with the AG pretreatment. NMR analysis revealed that recovered lignin was modified by glycerol through etherification of β-aryl ethers and esterification of hydroxycinnamic acids, which prevented lignin condensation and led to the generation of β-O-4 linkage-rich lignin at mild conditions (110 °C for 3 h and 5 h). This study suggests that crude glycerol is a suitable low-cost solvent for mild fractionation of lignocellulosic biomass into fermentable sugars and high-quality lignin for value-adding applications.