Technical Abstract: Marginal croplands, such as those in the Conservation Reserve Program (CRP), have been suggested as a source of biomass for biofuel production. However, little is known about the composition of plant species on these conservation grasslands or their potential for ethanol production. Our objective was to assess the potential of CRP and other conservation grasslands for biofuel production, describing the relationships of plant species richness and tall C4 prairie grass abundance with plant chemical composition and the resulting potential ethanol yield. We determined plant species composition and diversity at multiple scales with the modified Whittaker plot technique, aboveground biomass, plant chemical composition, and potential ethanol yield on conservation grasslands located over the major ecological regions of the northeastern USA. Conservation grasslands with higher numbers of plant species had lower biomass yields, and ethanol yield per unit biomass decreased as the number of plant species increased. Combining both observations led to a 77% decrease in biofuel yield per unit land area. In contrast, we found that as tall C4 prairie grass abundance increased, the number of plant species decreased, and aboveground biomass per unit land area and ethanol yield per unit biomass increased, resulting in over a 500% increased biofuel yield per unit land area. Our results suggest that addition of tall C4 prairie grasses on conservation grasslands led to higher biomass yields, to lower total plant species numbers, and to cellulose content that may lead to higher yield of cellulosic ethanol.