The resource efficiency of biofuel production via biomass pyrolysis is evaluated using exergy as an assessment metric. Three feedstocks, important to various sectors of U.S. agriculture, switchgrass, forest residue, and equine waste, are considered for conversion to bio-oil (pyrolysis oil) via fast pyrolysis, a process that has been identified as adaptable to on- or near-farm application. Biomass and biofuel production pathways are defined, material flows are determined, and exergy in- and outflows associated with biomass production and conversion are computed, including the depletion of exergy from its natural state (cumulative exergy demand, CExD). Sources of exergy depletion are quantified and categorized by energy carriers, e.g., electricity and diesel fuel, and materials, e.g., fertilizer, as well as renewable and nonrenewable resources. Yields for biomass to bio-oil conversion by fast pyrolysis are determined experimentally. Breeding factors, a measure of exergy production (the ratio of the chemical exergy of the output product to process exergy inputs), are determined for the production of biomass and bio-oil. The quantification of exergy depletion for process pathways enables the possible identification of more sustainable (resource efficient) pathways for biomass and bio-oil production. It is shown, for example, that feedstocks grown primarily for biomass such as switchgrass may be less sustainable using the exergy measure compared to use of residue (e.g., forest thinnings) or waste biomass (e.g., equine waste). With regard to the pyrolysis process, there is substantial reduction in exergy depletion when the coproducts noncondensable gases and biochar are recycled and utilized as a source of heat. The sustainability of biomass production and conversion, as measured by exergy depletion, is strongly influenced by energy carriers. The study reveals that the method of electricity production, i.e., on-site generation or grid electricity, as well as the choice of grid electricity can have a significant impact on sustainability. The exergy content of the bio-oil produced varies from 24 to 27 MJ/kg bio-oil, which is much lower than traditional fuels. However, the cumulative exergy depletion for the production and conversion to bio-oil varies from approximately 4 to 11 MJ/kg bio-oil, which is also much lower than traditional fuels. Breeding factors for biomass production and conversion to bio-oil based on cumulative exergy depletion vary from approximately 2 to 5, demonstrating the potential exergy benefit of bio-oil production using fast pyrolysis.