A ‘well-to-wheel’ analysis was conducted for bioethanol obtained from alfalfa stems by means of the Life Cycle Assessment (LCA) methodology. This analysis was compared with two blends of conventional gasoline with bioethanol (E10 and E85), all used in a mid-size car. A biochemical process including enzymatic hydrolysis and simultaneous saccharification and fermentation was considered. The life cycles of the fuels include gasoline production, alfalfa agriculture, lignocellulosic bioethanol production, blend production, and finally the use of fuels. The production of the alfalfa plant has two products: high-protein leaves for animal feed (the main driving force) and high-fiber stems. In this study, we assumed two allocation procedures based on mass and protein content, the latter reflecting the greater value of the leaves.According to the results, the use of bioethanol-based fuels leads to reduced global warming potential. A reduction in fossil fuel extraction of up to 72% could be achieved when pure bioethanol is used as transport fuel. On the contrary, bioethanol fuels are not the most suitable option when assessing acidification, eutrophication, and photochemical oxidant formation impact categories, mainly due to the higher impact from the upstream processes (specifically agricultural activities).LCA methodology helped to identify the key areas in the bioethanol production where researchers and technicians need to work to improve the environmental performance, paying special attention to enzyme production, onsite energy generation and distillation processes as well as agricultural activities. Copyright © 2010 Society of Chemical Industry and John Wiley & Sons, Ltd