The production of six regionally important cellulosic biomass feedstocks, including pine, eucalyptus, unmanaged hardwoods, forest residues, switchgrass, and sweet sorghum, was analyzed using consistent life cycle methodologies and system boundaries to identify feedstocks with the lowest cost and environmental impacts. Supply chain analysis was performed for each feedstock, calculating costs and supply requirements for the production of 453,592 dry tonnes of biomass per year. Cradle-to-gate environmental impacts from these modeled supply systems were quantified for nine mid-point indicators using SimaPro 7.2 LCA software. Conversion of grassland to managed forest for bioenergy resulted in large reductions in GHG emissions due to carbon uptake associated with direct land use change. By contrast, converting forests to cropland resulted in large increases in GHG emissions. Production of forest-based feedstocks for biofuels resulted in lower delivered cost, lower greenhouse gas (GHG) emissions, and lower overall environmental impacts than the agricultural feedstocks studied. Forest residues had the lowest environmental impact and delivered cost per dry tonne. Using forest-based biomass feedstocks instead of agricultural feedstocks would result in lower cradle-to-gate environmental impacts and delivered biomass costs for biofuel production in the southern U.S.