Fuels developed from plant cellulosic biomass offer a renewable and clean alternative to conventional fossil-based energy sources and contribute to the national and global energy security. Populus has become a model bioenergy crop. There are, however, biological and technological barriers that need to be overcome to achieve cost-effective production of biofuels using Populus. In particular, the key regulators of plant biomass recalcitrance have remained largely unknown. We took a forward genetics approach by screening an activation tagged Populus population for transgenic lines with altered phenotypic traits of wood chemistry and sugar release. We have identified a broad range of regulators of biomass recalcitrance, including phosphoglyceride transfer proteins, acetyltransferases, sugar transporters, protein kinases, transcription factors as well as unknown proteins. Interestingly, many of these regulators would not be thought to have any obvious role in cell wall biology. Molecular and biochemical characterization of these regulators will provide new insights into the mechanism of biomass recalcitrance and facilitate the selection and production of desired Populus feedstocks for biofuel production.