Expansion of bioenergy production is part of a global effort to reduce greenhouse gas emissions and mitigate climate change. Dedicated biomass crops will compete with other land uses as most high quality arable land is already used for agriculture, urban development, and biodiversity conservation. First, we explore the trade-offs between converting land enrolled in the U.S. Conservation Reserve Program (CRP) to switchgrass for biofuel production or preserving it for biodiversity. Next, we examine the trade-offs between agriculture, biodiversity, and biofuel across the central and eastern U.S. We compiled measures of biodiversity, agriculture, and biofuel from land cover classifications, species range maps, and mechanistic model output of switchgrass yield. We used a spatially-explicit optimization algorithm to analyze the impacts of small-to-large scale biomass production by identifying locations that maximize biofuel produced from switchgrass and minimize negative impacts on biodiversity and agriculture. Using CRP land for switchgrass production increases the land area required to meet biomass goals and the species range area altered for birds, amphibians, mammals, and reptiles. When conversion is not limited to CRP, conversion scenarios including biodiversity and agriculture trade-offs require greater than 100 % more area for switchgrass to reach the same production goals. When land conversion scenarios do not include biodiversity, twice the range area for reptiles and amphibians could be altered. Land-use trade-offs between biofuel production, agriculture, and biodiversity exist and alter optimum location of land conversion for low-to-high biofuel levels. This highlights the need for systematic land-use planning for the future.