Hydrogen produced by microorganisms is a topic of growing interest because of its potential for derivation from several agro-industrial by-products. In this study, we evaluated the hydrogen production of strains of genus Clostridium (Clostridium acetobutylicum and Clostridium butyricum) using glycerol as a carbon source. Fermentation studies were conducted using three initial concentrations of glycerol: 10, 30 and 50 g/L. The micro-organism growth kinetics and the amounts of solvents and gases were recorded over 48 h. The strain C. acetobutylicum exhibited the best results in terms of hydrogen production, the highest production yield (Y p/s) of 0.37 mol H2/mol glycerol and the highest level of productivity (0.75 mg H2/(L·h)). Based on these results, it is reasonable to conclude that glycerol could be effectively exploited as a carbon source for hydrogen production, which adds value to this primary by-product of standard biodiesel processes.