The increasing cost of fossil fuels such as petroleum, and a desire to curtail greenhouse gas emissions are driving the expansion of bioenergy. Plant biomass, including woody crops and grasses, are potential energy sources. We examined the biomass production, chemical composition, and energy content of selected hybrid poplar commercial clones such as NM6 (Populus nigra × Populus maximowiczii), D105 (Populus deltoides) and DN34 (P. deltoides × P. nigra) and native grasses and forbs established in polyculture systems in Minnesota, USA. In our study, we found that at the end of the 13-year growing season, NM6 had significantly greater (P = 0.0122) biomass production than D105 and DN34 with a total biomass production of 11.46 Mg ha−1. The chemical composition (mass fraction % on dry basis) of hybrid poplar clones generally contained 39% cellulose, 21% hemicellulose, 27% lignin, 1.3% ash content, and about 17,900–18,031 kJ kg−1 of dry wood. In contrast, after 7 years of growth, biomass such as that from the 5 grass mixture (5G), produced the highest amount of biomass (7.9 Mg ha−1) and largest theoretical ethanol yield (425 L Mg−1 of dry biomass), and contained mass fraction of 36% cellulose, 28% hemicellulose, 20% lignin, 6.04% ash content and about 16,731 kJ kg−1 of dry grass. Our data also showed that the slash left on site constituted a significant source of biomass (theoretically 375–390 L of ethanol for every megagram of biomass) that could be utilized for bioenergy. Seasonal timing of native grasses harvest significantly affected ethanol yield (P = 0.020) and energy content (P = 0.020). Strips of native grasses harvested in Spring 2009 that were allowed to re-grow and harvested in Fall 2009 showed greater potential for ethanol production than those harvested in Spring 2009. Thus, we suggest that hybrid poplars and native perennial grasses offer promising potential as alternative sources of renewable energy. Results of our study indicate that low-input high diversity systems can be utilized as an alternative source of biomass for energy and it could facilitate commercial production of the crops.