Biomass is a potential energy resource that can be converted to hydrogen and is available from a wide range of sources. In this study, non-conventional biomass sources: soybean (Glycine max.), cassava (Manihot esculenta) and banana rachis (Musa paradisiaca L.) had been tested for hydrogen production from biomass pyrolysis. The influence of biomass microstructure and reducing sugar content on the hydrogen production was studied through a high-energy ball-milling pre-process of the soybean source. The results indicate that the maximum production of hydrogen was obtained at 275°C independently of the biomass source. The biomass microstructure and reducing sugar content does not seem to have an effect on hydrogen production from biomass pyrolysis. Soybean thermal stability is increased during the ball-milling process. High-energy ball-milling can be a good, easy and low cost method to increase biomass reducing sugar content.