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Perennial biomass from grasslands managed for conservation of soil and biodiversity can be harvested for bioenergy. Until now, the quantity and quality of harvestable biomass from conservation grasslands in Minnesota, USA, was not known, and the factors that affect bioenergy potential from these systems have not been identified. We measured biomass yield, theoretical ethanol conversion efficiency, and plant tissue nitrogen (N) as metrics of bioenergy potential from mixed-species conservation grasslands harvested with commercial-scale equipment. With three years of data, we used mixed-effects models to determine factors that influence bioenergy potential. Sixty conservation grassland plots, each about 8 ha in size, were distributed among three locations in Minnesota. Harvest treatments were applied annually in autumn as a completely randomized block design. Biomass yield ranged from 0.5 to 5.7 Mg ha−1. May precipitation increased biomass yield while precipitation in all other growing season months showed no affect. Averaged across all locations and years, theoretical ethanol conversion efficiency was 450 l Mg−1 and the concentration of plant N was 7.1 g kg−1, both similar to dedicated herbaceous bioenergy crops such as switchgrass. Biomass yield did not decline in the second or third year of harvest. Across years, biomass yields fluctuated 23% around the average. Surprisingly, forb cover was a better predictor of biomass yield than warm-season grass with a positive correlation with biomass yield in the south and a negative correlation at other locations. Variation in land ethanol yield was almost exclusively due to variation in biomass yield rather than biomass quality; therefore, efforts to increase biomass yield might be more economical than altering biomass composition when managing conservation grasslands for ethanol production. Our measurements of bioenergy potential, and the factors that control it, can serve as parameters for assessing the economic viability of harvesting conservation grasslands for bioenergy. [ABSTRACT FROM AUTHOR]