A system has been proposed using alfalfa (Medicago sativa L.) as a biofuel feedstock, where the stems would be processed to produce energy and the leaves used as a livestock feed. Our objectives were to evaluate the effects and interactions of environment, population density, and harvest maturity on leaf and stem yield of alfalfa germplasms differing in fall dormancy and leaf/stem ratio. Four alfalfa germplasms established at four population densities (450, 180, 50, and 16 plants m-2) were harvested at the early bud and green pod maturity stages and evaluated in three environments for leaf and stem yield. All main effects and several two-way interactions influenced leaf and stem yield (P < 0.01). The population density x harvest maturity interaction had the greatest impact on yield. Leaf and stem yield per unit area increased as population density increased from 16 to 450 plants m-2 at the early bud stage. In contrast, leaf and stem yield increased as population density increased from 16 to 180 plants m-2, but decreased dramatically at 450 plants m-2 at the green pod maturity stage. Delaying harvest until the green pod maturity stage and decreasing population density to 180 plant m-2 maximized both leaf and stem yield in all four alfalfa germplasms studied. Decreasing population density to 180 plants m-2, and harvesting twice per season at a later maturity stage would be a effective management strategy for maximizing yield in an alfalfa biomass energy or biofuel production system.