Clostridium thermocellum is a model microorganism for consolidated bioprocessing (CBP) of biomass to fuels and chemicals. One of the challenges for industrial application of this organism is its low native ethanol tolerance (~ 2% w/v) as production concentrations of >50g/L ethanol are required for economical product recovery. In this study, we report the development and characterization of mutant strains, derived from C. thermocellum ATCC 27405, with increased tolerance to ethanol, up to 50 g/l. Using a combination of genome resequencing and multiple omics techniques, we have characterized the genomic changes in the ethanol tolerant mutant strains and examined their physiological response in ethanol stress studies. Data will be presented on fermentation studies, conducted using 27405 and mutant strains growing on 5 g/L Avicel or cellobiose, challenged during growth with 10 g/L or 40 g/L final concentration of ethanol. Samples taken during the course of the fermentations were analyzed using a combination of transcriptomics, metabolomics, and proteomics methods to evaluate the short- and long-term cellular responses to added ethanol. The results of this combined omics analyses regarding genes involved in ethanol tolerance and the potential for development of improved C. thermocellum strains for industrial biofuel production will be discussed.