The aim of this work was to evaluate the influence of partial replacement of maize flour by sub-valuated whole grain flours on the interactions and spatial distribution of the main components of extruded snacks. Non-destructive, fast spectroscopic and microscopic complementary tools were employed. All blends (composed by maize + 25% of pearl millet, red sorghum, quinoa or hairless canary seed) presented lower carbohydrate and lipid contents and higher protein, ash and dietary fiber levels than the control maize extrudates. These compositional changes were reflected in FT-MIR and FT-Raman spectra which also allowed detecting modifications at molecular level, such as protein agglomeration, amylose-lipids complexes formation and surface exposure of carotenoids. Tridimensional distribution and spatial arrangements of the main components in the extruded samples were studied through CLSM and XPS. These properties are related to quality aspects such as lipid oxidation sensitivity and textural properties. The combined FT-MIR, FT-Raman, CLSM and XPS were helpful tools to understand thermo-mechanical modifications of starch, proteins and lipids as a consequence of the extrusion process.