This study investigated the structural recalcitrance of holocellulose from lignocellulosic biomasses (LCBs): sugarcane-bagasse (R-SCB), R-Poplar, and R-Spruce, considering their heterogeneous recalcitrance. Acidic sodium chlorite (ASC) pretreatment was employed for different time intervals (0-4 h) and extracted holocellulose samples (SCB, poplar, and spruce) with comparable lignin content (~7 %) were further alkaline-incubated (1-SCB, 1-Poplar, and 1-Spruce) with 1 % NaOH at 50 °C for 1 h. Varying enzymatic hydrolysis (EH) results indicate distinct structural heterogeneity, which was further investigated through the deconvolution of FTIR, XRD, XPS, and 13CP MAS NMR spectra, highlighting the synergistic interplay among various allomorphs of cellulose that determine the glucose yield. Results indicated that SCB and 1-SCB exhibited superior structural characteristics, with an interplay of reduced intermolecular hydrogen bonding, increased intramolecular bonding and oxidized surface. Moreover, alkaline incubation significantly hydrolyzed the lignin-carbohydrate complexes (LCCs) from holocellulose, such as benzyl ether (BE) and phenyl glycoside (PG), indicating the removal of the xylan-lignin matrix from holocellulose. This ultimately reduces steric hindrance and hydrophobicity by hydrolyzing recalcitrant lignin and LCCs, thereby disrupting the LCB matrix for more efficient LCB utilization. This study provides detailed insights into LCB structural disruption, guiding the pretreatment optimization processes for more efficient biorefinery applications.