Abstract: Abstract Chemogenomics, the study of genomic responses to chemical compounds, has the potential to elucidate the basis of cellular resistance to those chemicals. This knowledge can be applied to improve the performance of strains of industrial interest. In this study, a collection of approximately 5,000 haploid single deletion mutants of Saccharomyces cerevisiae in which each nonessential yeast gene was individually deleted, was screened for strains with increased susceptibility toward stress induced by high-glucose concentration (30% w/v), one of the main stresses occurring during industrial alcoholic fermentation processes aiming the production of alcoholic beverages or bio-ethanol. Forty-four determinants of resistance to high-glucose stress were identified. The most significant Gene Ontology (GO) terms enriched in this dataset are vacuolar organization, late endosome to vacuole transport, and regulation of transcription. Clustering the identified resistance determinants by their known physical and genetic interactions further highlighted the importance of nutrient metabolism control in this context. A concentration of 30% (w/v) of glucose was found to perturb vacuolar function, by reducing cell ability to maintain the physiological acidification of the vacuolar lumen. This stress also affects the active rate of proton efflux through the plasma membrane. Based on results of published studies, the present work revealed shared determinants of yeast resistance to high-glucose and ethanol stresses, including genes involved in vacuolar function, cell wall biogenesis (ANP1), and in the transcriptional control of nutrient metabolism (GCN4 and GCR1), with possible impact on the design of more robust strains to be used in industrial alcoholic fermentation processes.