Lignocellulosic biomass is an important renewable feedstock with high potential to develop new processes of biobased products in the fields of chemistry, energy and materials. However, it is also highly recalcitrant to transformation and a pretreatment step is required to facilitate its conversion. In this study, steam-explosion pretreatment was applied to two wood species of economic relevance, spruce and beechwood samples to highlight its impact on the structure and composition of wood with the aim to predict biomass reactivity to optimize its conversion into biobased materials and decarbonized hydrogen. Several severities of pretreatment were studied by varying the temperature and the time of residence and by combining or not steam-explosion with diluted acid. The impact of the pretreatment was analysed by implementing a multimodal approach at different scales, from biomass composition to cell morphology by combining chemical analyses, spectroscopy, colorimetry and macroscopic imaging. Particularly, an automated method was developed to quantify fluorescence and cell morphological characteristics changes following steam explosion pretreatment based on images acquired by fluorescence macroscopy. Results in fluorescence macroscopy showed the impact of steam-explosion on softwood and hardwood, as fluorescence intensity of samples was highly altered after steam-explosion pretreatment, especially for the most severe. Morphological changes were also revealed: shrinkage of cells and deformation of cell walls manifested as the loss of rectangularity or circular shape, for tracheids in spruce and vessels in beechwood respectively. Quantification of morphological parameters related to cell lumens showed there was a correlation with the severity of pretreatment. It was also possible to relate fluorescence intensity and morphological parameters to compositional and colorimetric data. This multimodal characterization of softwood and hardwood after steam-explosion pretreatment helps towards a better understanding of the effect of pretreatment on biomass recalcitrance. In addition, integrating chemical, spectroscopic and morphological features would allow ranking markers of the lignocellulose recalcitrance regarding cellulose accessibility and ability to convert into biobased products.