Two primary approaches to perennial biofuel crop production studied so far are fertilized grassmonocultures and low-input high-diversity grasslands. While high-yielding perennial grass varieties arebeing developed in fertilized monocultures, breeding for yield in low-input high-diversity systemswould be difficult. Before initiating breeding for low-input systems, it is therefore important to know theminimum number of functional groups and species required for maximum biomass harvest from lowinputgrasslands. We controlled the number of perennial grassland species in 168 plots in Minnesota,USA. Species were selected at random from a pool of 18, and 1, 2, 4, 8, or 16 were planted in each plot.Aboveground biomass was measured annually, and the plots were burned each spring. We found astrongly positive log-linear relationship between average annual aboveground biomass and plantedspecies number, but a large proportion of plot-to-plot variability remained unexplained. Weperformed aconditional analysis of the aboveground biomass data to determine whether considering species identitywould reduce the minimum number of species necessary in order to achieve yields similar to the highestdiversity treatments. A model that accounted for the presence of legumes in general, and for the presenceof the legume species Lupinus perennis in particular, showed no increase in biomass yield with increasedspecies number. Over 11 years, average yields of L. perennis/C4 grass bicultures were similar to those of16-species (maximum diversity) plots, and both were >200% greater than the average of monocultures.Thus, under low-input conditions, the choice of the appropriate few perennial plant species for eachlocation might result in systems with biomass yields similar to those from high-diversity systems.Because breeding biofuel crops in diverse mixtures would introduce complexity that is unwarranted interms of maximum biomass yield, the first biofuel crop breeding programs for low-input systems arelikely to accelerate progress by focusing on grass–legume bicultures.