Biomass pyrolysis by solid acid catalysts is one of many promising technologies for sustainable production of hydrocarbon liquid fuels and value-added chemicals, but these complex chemical transformations are still poorly understood. A series of well-defined model SiO2-supported alumina catalysts were synthesized and molecularly characterized, under dehydrated conditions and during biomass pyrolysis, with the aim of establishing fundamental catalyst structure–activity/selectivity relationships. The nature and corresponding acidity of the supported AlOx nanostructures on SiO2 were determined with 27Al/1H NMR and IR spectroscopy of chemisorbed CO, and DFT calculations. Operando time-resolved IR–Raman–MS spectroscopy studies revealed the molecular transformations taking place during biomass pyrolysis. The molecular transformations during biomass pyrolysis depended on both the domain size of the AlOx cluster and molecular nature of the biomass feedstock. These new insights allowed the establishment of fundamental structure–activity/selectivity relationships during biomass pyrolysis.