Among the various sources of biomass, the lignocellulosic sources such as agricultural residue, dedicated energy grasses, forestry residue and short rotation woody biomass are expected to satisfy a significant fraction of the total future demand. However, while technologically feasible, the production of biofuels from lignocellulosic biomass is not techno-economically feasible. The presence of various uncertainties creates a number of challenges in overcoming these hurdles. The biomass to biofuel system is impacted by variability, both natural as well as anthropogenic, at almost all stages. This includes variability in feedstock production on a seasonal basis, variability in feedstock composition, variability in process efficiencies and variability in prices of raw material and products. This makes the optimization of the complete system extremely challenging. This article highlights the presence of these uncertainties and their impact on the operations of the biomass to biofuel systems. A detailed classification of uncertainties in biomass to biofuel systems, methods of incorporating uncertainties in process optimization and supply chain design are reviewed and explained. The paper will also review the work done to address these uncertainties using systematic methods.