Lignocellulose biomass can be transformed into sustainable chemicals, materials and energy to contribute to bioeconomy expansion. Due to the feedstock variety and natural recalcitrance to fractionation, transformation processes such as pretreatments and hydrolysis of lignocellulose must be adapted to biomass species and desired final products. That is why predicting reactivity of biomass from some ab initio properties would be advantageous to save time. We have used Raman spectroscopy as a spectral technique to characterize poplar wood samples before and after dilute acid pretreatement (DAP). Indeed, confocal Raman microscopy can provide information about the concentration and the spatial distribution of chemical components in cell walls. Changes in the spatial distribution of polymers in the cell walls of pretreated poplar could thus be investigated in situ. The average Raman spectra of untreated and pretreated poplar were collected from various cell wall regions: cell corner, compound middle lamella, and secondary wall. This topochemical analysis revealed that modifications in chemical composition after DAP are heterogeneous throughout the poplar cell walls and stronger with higher DAP severity. Interestingly, Raman data obtained from specific ratio between lignin and cellulose-related bands, which is directly related to polymer accessibility, showed very strong Pearson correlations (> 0.93) with enzymatic hydrolysis of pretreated samples. Such markers which are easy to measure can thus be considered as excellent predictors of poplar hydrolysis.