Lignocellulosic substrates derived from waste biomass have become an attractive feedstock for the production of inexpensive, more environmentally-friendly biofuels.  For example, spent sulfite liquor (SSL), a carbohydrate-rich effluent produced in sulfite pulping, can be used to add value to the pulp and paper industry by using the sugars it contains to produce ethanol.  However, using SSL in such a capacity requires a robust, ethanologenic microorganism that can withstand the substrate toxicity that is due to the presence of inhibitory compounds like furfural, 5-hydroxymethylfurfural (HMF) and acetic acid.  Saccharomyces cerevisiae is currently used for the production of ethanol from SSL.  This industrially well-established yeast, though a robust starting organism for SSL fermentation, will still succumb to toxicity and inhibition, especially in the most inhibitor rich forms of SSL such as hardwood SSL (HWSSL).  To establish a S. cerevisiae strain that can overcome such a complex and incompletely understood form of inhibitory pressure, a genome shuffling method was developed to create a better SSL fermenter.  This method aims to improve polygenic traits by generating a pool of mutants with improved phenotypes, followed by iterative recombination between their genomes.  Through five rounds of shuffling and screening, three strains were obtained that are able to not only survive in HWSSL, but grow to a limited extent.  Our results show that the tolerance of these strains to SSL translates into an increased capacity to produce ethanol over time using this substrate, due to continued viability of the yeast population.