Biofuels can reduce greenhouse gas (GHG) emissions by replacing fossil fuels. However, the energy yield from agronomic crops varies due to local climate, weather and soil variability. A variation in the yield of raw material used (feedstock) could also cause variability in GHG reductions if biofuels are used. The goal of the present study was to determine the net reduction of GHG emissions if ethanol from wheat produced in different regions of the south-eastern USA is used as an alternative to gasoline from fossil fuel sources. Two scenarios were investigated; the first included ethanol produced from grain only, and the second included ethanol produced from both grain and wheat straw. Winter wheat yield was simulated with the Cropping System Model (CSM)-CERES-Wheat model for climate, soil and crop management representing six counties in the following USA states: Alabama, Florida and Georgia. Ethanol production was determined from the simulated grain and straw yields together with fixed grain and straw yield ethanol ratios. Subsequently, net reductions in GHG emissions were determined by accounting for the emissions from the replaced gasoline, and by animal feed and electricity that were replaced by ethanol processing co-products. Greenhouse gases that were emitted in the ethanol production chain were also taken into account. Across all locations, the reduction in GHG emissions was 187 g CO2-equivalents/km in the grain-only scenario and 208 g CO2-equivalents/km in the grain and straw scenario. The reductions in GHG emissions varied significantly between locations and growing seasons within the two scenarios. Similar approaches could be applied to assess the environmental impact of GHG emissions from other biofuels.