Biofuels account for 1–2% of global transportation fuel and their share is projected to continue rising, with potentially serious consequences for water resources. However, current literature does not present sufficient spatial resolution to characterize this localized effect. We used a coupled agro-climatic and life cycle assessment model to estimate the water resource impacts of bioenergy expansion scenarios at a county-level resolution. The study focused on the case of California, with its range of agroecological conditions, water scarcity, and aggressive alternative fuel incentive policies. Life cycle water consumption for ethanol production in California is up to 1000 times that of gasoline due to a cultivation phase that consumes over 99% of life cycle water use for agricultural biofuels. This consumption varies by up to 60% among different feedstocks and by over 350% across regions in California. Rigorous policy analysis requires spatially resolved modeling of water resource impacts and careful consideration of the various metrics that might act to constrain technology and policy options.