Ethanol tolerance in Saccharomyces cerevisiae is a complex polygenic trait. As a toxin, ethanol damages multiple cell constituents as well as being both a substrate and product of the metabolism of S.cerevisiae. This complexity has made ethanol tolerance difficult to study. Deletion screens have identified hundreds of genes that impair ethanol tolerance by their absence and hence might help survival in high ethanol environments. Similarly, expression studies have revealed genes that respond to ethanol shock, but it is unclear whether those genes are likely targets for improvement of ethanol tolerance in strains growing normally. In addition, those yeasts that are currently commercially exploited for their high ethanol tolerance in the brewing and bioethanol industries are commonly aneuploid or polyploid which makes it difficult to correlate particular features of their genotype with the ethanol tolerant phenotype.Experimental evolution can reveal genetic changes that change competitive fitness. It is practical to run numerous competitions in parallel between isogenic S.cerevisiae strains for hundreds of generations under ethanol stress, after which whole genome sequencing can identify the genetic changes. Fluorescent tagging of those strains can reveal small changes in population dynamics.We propagated 144 populations in batch culture for between 100 and 200 generations under 4 ethanol regimes (0%, 4.5%, 6.5% and ramped 0-10%). We monitored the progress of evolution by using mixtures of two fluorescently tagged, but otherwise isogenic, haploid, hoDelta (site-specific endonuclease deletant) founder strains (DeLuna et al 2008). Population dynamics measured using fluorescently labelled strains indicated that changes had occurred in competitive fitness due to adaptive evolution. Cell-size measurement and flow cytometry showed that evolved populations were diploid or triploid and the transition to higher ploidy occurred more rapidly with increasing ethanol stress. During the experimental evolution three strains evolved the capacity to grow on organic acids. We have sequenced the complete genomes of eight evolved strains. These strains are confirmed as being diploid, but not aneuploid. Sequencing of evolved strains revealed mutations that have not been previously characterised in deletion or expression studies of ethanol or organic acid tolerance in S.cerevisiae.Both increasing ploidy, to produce triploids as well as diploids, and the acquisition of organic acid tolerance under ethanol stress are unexpected outcomes that have implications for future work.