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Biofuels derived from low-input high-diversity (LIHD) mixtures of native grassland perennials can provide more usable energy, greater greenhouse gas reductions, and less agrichemical pollution per hectare than can corn grain ethanol or soybean biodiesel. High-diversity grasslands had increasingly higher bioenergy yields that were 238% greater than monoculture yields after a decade. LIHD biofuels are carbon negative because net ecosystem carbon dioxide sequestration (4.4 megagram hectare–1 year–1 of carbon dioxide in soil and roots) exceeds fossil carbon dioxide release during biofuel production (0.32 megagram hectare–1 year–1). Moreover, LIHD biofuels can be produced on agriculturally degraded lands and thus need to neither displace food production nor cause loss of biodiversity via habitat destruction.