A spatially explicit life cycle water analysis framework is proposed, in which a standardized water footprint methodology is coupled with hydrologic modeling to assess blue water, green water (rainfall), and agricultural grey water discharge in the production of biofuel feedstock at county-level resolution. Grey water is simulated via SWAT, a watershed model. Evapotranspiration (ET) estimates generated with the Penman-Monteith equation and crop parameters were verified by using remote sensing results, a satellite-imagery-derived data set, and other field measurements. Crop irrigation survey data are used to corroborate the estimate of irrigation ET. An application of the concept is presented in a case study for corn-stover-based ethanol grown in Iowa (United States) within the Upper Mississippi River basin. Results show vast spatial variations in the water footprint of stover ethanol from county to county. Producing 1 L of ethanol from corn stover growing in the Iowa counties studied requires from 4.6 to 13.1 L of blue water (with an average of 5.4 L), a majority (86%) of which is consumed in the biorefinery. The county-level green water (rainfall) footprint ranges from 760 to 1000 L L−1. The grey water footprint varies considerably, ranging from 44 to 1579 L, a 35-fold difference, with a county average of 518 L. This framework can be a useful tool for watershed- or county-level biofuel sustainability metric analysis to address the heterogeneity of the water footprint for biofuels.