A major bottleneck in the conversion of lignocellulosic biomass into ethanol by the brewer’s yeast Saccharomyces cerevisiae is the inhibition of fermentation resulting from cellular stress, which can be caused by degradation products generated during feedstock pretreatment.  Our ongoing research at the Great Lakes Bioenergy Research Center has compared environmental stress tolerance between laboratory and wild yeast strains, and discovered that genetic background is a significant determinant of tolerance.  This also suggested that S. cerevisiae strains isolated from diverse natural or industrial environments may have traits that allow for greater tolerance to toxins generated from pretreatment of lignocellulosic biomass.  We sought to identify environmental and industrial S. cerevisiae isolates with greater tolerance to these stresses than laboratory strains, and understand the genetic, biochemical, and molecular traits contributing to these phenotypic differences.  Thus, we examined the growth properties of hundreds of diverse S. cerevisiae strains cultured in various lignocellulosic hydrolysates.  These various hydrolysates were prepared from biomass pretreated by ammonium fiber expansion, ionic liquid, or dilute acids.  High throughput robotic screening and computational methods were employed to identify the top performing strains.  One of the top strains included an environmental isolate that, in contrast to most other strains, grows well in AFEX-pretreated corn stover hydrolysate at elevated temperatures of 37 and 40°C.  Further characterization of these strains for ethanol production and ability to ferment xylose is currently in progress.  Results from these studies will provide insight for improving existing production strains, or developing new stress tolerant strains for cellulosic ethanol production.