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Saccharomyces cerevisiae is an important unicellular yeast species within the biotechnological and the food and beverage industries. A significant application of this species is the production of ethanol, where concentrations are limited by cellular toxicity, often at the level of the cell membrane. Here, we characterize 61 S. cerevisiae strains for ethanol tolerance and further analyze five representatives with various ethanol tolerances. The most tolerant strain, AJ4, was dominant in coculture at 0 and 10% ethanol. Unexpectedly, although it does not have the highest noninhibitory concentration or MIC, MY29 was the dominant strain in coculture at 6% ethanol, which may be linked to differences in its basal lipidome. Although relatively few lipidomic differences were observed between strains, a significantly higher phosphatidylethanolamine concentration was observed in the least tolerant strain, MY26, at 0 and 6% ethanol compared to the other strains that became more similar at 10%, indicating potential involvement of this lipid with ethanol sensitivity. Our findings reveal that AJ4 is best able to adapt its membrane to become more fluid in the presence of ethanol and that lipid extracts from AJ4 also form the most permeable membranes. Furthermore, MY26 is least able to modulate fluidity in response to ethanol, and membranes formed from extracted lipids are least leaky at physiological ethanol concentrations. Overall, these results reveal a potential mechanism of ethanol tolerance and suggest a limited set of membrane compositions that diverse yeast species use to achieve this. IMPORTANCE Many microbial processes are not implemented at the industrial level because the product yield is poorer and more expensive than can be achieved by chemical synthesis. It is well established that microbes show stress responses during bioprocessing, and one reason for poor product output from cell factories is production conditions that are ultimately toxic to the cells. During fermentative processes, yeast cells encounter culture media with a high sugar content, which is later transformed into high ethanol concentrations. Thus, ethanol toxicity is one of the major stresses in traditional and more recent biotechnological processes. We have performed a multilayer phenotypic and lipidomic characterization of a large number of industrial and environmental strains of Saccharomyces to identify key resistant and nonresistant isolates for future applications.