Life-cycle analysis (LCA) is an important tool used to assess the energy and environmental impacts of biofuels. Here, we review biofuel LCA methodology and its application in transportation fuel regulations in the United States, the European Union, and the United Kingdom. We examine the application of LCA to the production of ethanol from corn, sugarcane, corn stover, switchgrass, and miscanthus. A discussion of methodological choices such as co-product handling techniques in biofuel LCA is also provided. Further, we discuss the estimation of greenhouse gas (GHG) emissions of land use changes (LUC) potentially caused by biofuels, which can significantly influence LCA results. Finally, we provide results from LCAs of ethanol from various sources. Regardless of feedstock, bioethanol offers reduced GHG emissions over fossil-derived gasoline, even when LUC GHG emissions are included. This is mainly caused by displacement of fossil carbon in gasoline with biogenic carbon in ethanol. Of the ethanol pathways examined, corn ethanol has the greatest life-cycle GHG emissions and offers 30% reduction in life-cycle GHG emissions as compared to gasoline when LUC GHG emissions are included. Miscanthus ethanol demonstrates the highest life-cycle GHG emissions reductions compared to gasoline, 109%, when LUC GHG emissions are included.