Biofuel production processes at high gravity are currently under development. Most of these processes however use sugars or first generation feedstocks as substrate. This paper presents the results of a life cycle assessment (LCA) of the production of bio-ethanol at high gravity conditions from a second generation feedstock, namely, wheat straw. The LCA used lab results of a set of 36 process configurations in which dry matter content, enzyme preparation and loading, and process strategy were varied. The LCA results show that higher dry matter content leads to a higher environmental impact of the ethanol production, but this can be compensated by reducing the impact of enzyme production and use, and by polyethylene glycol addition at high dry matter content. The results also show that the renewable and non-renewable energy use resulting from the different process configurations ultimately determine their environmental impact.