Legislation on biofuels production in the USA and Europe is directing food crops towards the production of grain-based ethanol, which can have detrimental consequences for soil carbon sequestration, nitrous oxide emissions, nitrate pollution, biodiversity and human health. An alternative is to grow lignocellulosic (cellulosic) crops on ‘marginal’ lands. Cellulosic feedstocks can have positive environmental outcomes and could make up a substantial proportion of future energy portfolios. However, the availability of marginal lands for cellulosic feedstock production, and the resulting greenhouse gas (GHG) emissions, remains uncertain. Here we evaluate the potential for marginal lands in ten Midwestern US states to produce sizeable amounts of biomass and concurrently mitigate GHG emissions. In a comparative assessment of six alternative cropping systems over 20 years, we found that successional herbaceous vegetation, once well established, has a direct GHG emissions mitigation capacity that rivals that of purpose-grown crops (−851 ± 46 grams of CO2 equivalent emissions per square metre per year (gCO2e m−2 yr−1)). If fertilized, these communities have the capacity to produce about 63 ± 5 gigajoules of ethanol energy per hectare per year. By contrast, an adjacent, no-till corn–soybean–wheat rotation produces on average 41 ± 1 gigajoules of biofuel energy per hectare per year and has a net direct mitigation capacity of −397 ± 32 gCO2e m−2 yr−1; a continuous corn rotation would probably produce about 62 ± 7 gigajoules of biofuel energy per hectare per year, with 13% less mitigation. We also perform quantitative modelling of successional vegetation on marginal lands in the region at a resolution of 0.4 hectares, constrained by the requirement that each modelled location be within 80 kilometres of a potential biorefinery. Our results suggest that such vegetation could produce about 21 gigalitres of ethanol per year from around 11 million hectares, or approximately 25 per cent of the 2022 target for cellulosic biofuel mandated by the US Energy Independence and Security Act of 2007, with no initial carbon debt nor the indirect land-use costs associated with food-based biofuels. Other regional-scale aspects of biofuel sustainability, such as water quality and biodiversity, await future study.