Ethanol, produced by S. cerevisiae fermentation, is the main biofuel used worldwide. The toxicity effect of ethanol on yeast, which inhibits industrial productivity, calls for better understanding of the genetic basis of ethanol tolerance. We used an Advanced Intercross Line design, Selective DNA Pooling and whole-genome sequencing, with LOESS smoothing and log-drop boundaries for high resolution mapping of QTLs affecting ethanol tolerance in the F6 of a cross between two widely separated S. cerevisiae strains.