Bioethanol is a renewable and clean energy source, highly valuable especially nowadays with climate change being at high awareness. Produced by the yeast S. cerevisiae, bioethanol is the main product of a carbon source fermentation. As the accumulated ethanol is toxic to the cells, high ethanol tolerance is a primary demand for industrial bioethanol yeast strains. Ethanol tolerance is a polygenic trait that has been previously mapped in our lab using a specific di-allelic strain platform. To expand this knowledge, we aimed at testing the effect of a larger allele panel at each of the main QTLs mapped on ethanol tolerance. Although S. cerevisiae is an extensively studied and an easily manipulated organism, almost no genetic engineering has been attempted on bioethanol yeast for the purpose of enhancing ethanol productivity. Here we present the attempt to use the Cas-9 protein, as a genome engineering tool for allele replacement, in two strains of yeast (a common laboratory strain and own-developed strain oriented for bioethanol production). Unfortunately, attempts to replace existing alleles in these strains by different ones using Cas-9 have failed to produce results. Therefore, a different methodology was used to assess the contribution of additional alleles to ethanol tolerance, based on genetic segregation of alleles in the F1 generation of a self-hybridized strain. We measured the growth ability under ethanol stress of 192 offspring and correlated them with segregating SNPs representing nine high influence QTLs. This represents the first step towards genomic based breeding to improve ethanol resistance and production.