Lignocellulosic biomass (LB) is a renewable feedstock which states as a promising alternative to fossil resources to produce biomolecules, biomaterials, and energy through biorefinery processes. However, the complex network formed by LB constituents leads to its recalcitrance and requires a pretreatment step to ensure optimal biological conversion. Continuous steam explosion (CSE) induces chemical and morphological alterations of LB fibers and allows deconstruction of lignocellulosic cell walls to facilitate access to cellulose. Three species of wood (oak, poplar, and spruce) were pretreated with pilot scale CSE at different severities by varying temperature and residence time. Glucose release after enzymatic hydrolysis and bioethanol production were monitored (Figure 1) and data we re modelled using an experimental design approach to determine optimal CSE conditions and propose different scenarios. Multimodal characterization of pretreated samples was also carried out to investigate the impact of chemical and physical modifications o n 2G sugars release and on bioethanol production and how they can help predicting LB reactivity for biological conversion. Some features such as particle size, chemical composition, and lignin modifications were found to be related to bioethanol production increase, while others such as crystallinity and hydrophobicity had a negative impact during enzymatic saccharification. Depending on the wood species, specific band of infrared spectrum or fluorescence intensity were found to be excellent proxies to pred ict saccharification rate and thus bioethanol production. Overall, our study demonstrates the importance of the choice of starting LB material and pretreatment conditions to maximize bioethanol production and the necessity of performing a multimodal approa ch to pinpoint the multiscale LB features having an impact on bioethanol production.