Switchgrass and Populus could provide energy-rich feedstocks for the consolidated bioprocessing of cellulosic biomass to ethanol. However, advances in overcoming the recalcitrance of converting plant cell walls to fermentable sugars are needed for an economically viable process. In order to investigate the diversity of microorganisms that can efficiently hydrolyze the cellulosic fraction of switchgrass and Populus at elevated temperatures, we collected water, sediment and decaying wood samples from thermal features located in Yellowstone National Park, Wyoming, USA. Enrichment cultures using acid pretreated switchgrass and Populus as the primary carbon and energy source supported the anaerobic growth of microorganisms at temperatures between 60 to 80 deg. C. Cloning and sequencing of amplified 16S rDNA from several enrichments verified the presence of known cellulolytic, extremely thermophilic bacteria including Caldicellulosiruptor and Thermoanaerobacter spp. Clones representing uncultivated clades of Dictyoglomus, Thermodesulfovibrio, and Nitrospira were also recovered. In order to characterize individual organisms in detail, a high-throughput (HT) isolation system based on flow cytometry was developed that allowed rapid separation and growth of cellulolytic, extreme thermophiles. Coupled to modern screening and phenotyping approaches, the HT isolation system will be used to assemble a large collection of novel cellulolytic, ethanol producing microorganisms from diverse environments.