Lignocellulosic biofuels can help fulfill escalating demands for liquid fuels and mitigate the environmental impacts of petroleum-derived fuels. Two key factors in the successful large-scale production of lignocellulosic biofuels are pretreatment (in biological conversion processes) and a consistent supply of feedstock. Cellulosic biomass tends to be bulky and difficult to handle, thereby exacerbating feedstock supply challenges. Currently, large biorefineries face many logistical problems because they are fully integrated, centralized facilities in which all units of the conversion process are present in a single location. The drawbacks of fully integrated biorefineries can potentially be dealt by a network of distributed processing facilities called ‘Regional Biomass Processing Depots’ (RBPDs) which procure, preprocess/pretreat, densify and deliver feedstock to the biorefinery and return by-products such as animal feed to end users. The primary objective of this study is to perform a comparative life cycle assessment (LCA) of distributed and centralized biomass processing systems. Additionally, we assess the effect that apportioning land area to different feedstocks within a landscape has on the energy yields and environmental impacts of the overall systems. To accomplish these objectives, we conducted comparative LCAs of distributed and centralized processing systems combined with farm-scale landscapes of varying acreages allocated to a ‘corn-system’ consisting of corn grain, stover and rye (grown as a winter double crop) and two perennial grasses, switchgrass and miscanthus. The distributed processing system yields practically the same total energy and generates 3.7% lower greenhouse gas emissions than the centralized system. Sensitivity analyses identified perennial grass yields, biomass densification and its corresponding energy requirements, transport energy requirements and carbon sequestration credits for conversion from annual to perennial crops as key parameters that significantly affect the overall results.