The introduction in modern breweries of tall cylindroconical fermentors, replacing the traditional open fermentation vats, unexpectedly revealed strong inhibition of flavor production by the high CO₂ pressure in the fermentors. We have screened our collection of Saccharomyces cerevisiae strains for strains displaying elevated tolerance to inhibition of flavor production by +0.65 bar CO₂, using a laboratory scale CO₂ pressurized fermentation system. We focused on the production of isoamyl acetate, a highly desirable flavor compound conferring fruity banana flavor in beer and other alcoholic beverages, from its precursor isoamyl alcohol (IAAc/Alc ratio). We selected the most tolerant Saccharomyces cerevisiae strain, saké yeast Kyokai no. 1, isolated a stable haploid segregant seg63 with the same high IAAc/Alc ratio under CO₂ pressure, crossed seg63 with the unrelated inferior strain ER7A and phenotyped 185 haploid segregants, of which 28 displaying a high IAAc/Alc ratio were pooled. Mapping of Quantitative Trait Loci (QTLs) by whole-genome sequence analysis based on SNP variant frequency revealed two QTLs. In the major QTL, reciprocal hemizygosity analysis identified MDS3 as the causative mutant gene, a putative member of the TOR signaling pathway. The MDS3^Seg.63 allele was dominant and contained a single causative point mutation, T2171C, resulting in the F274S substitution. Introduction of MDS3^Seg.63 in an industrial tetraploid lager yeast with CRISPR/Cas9 enhanced isoamyl acetate production by 145% under CO₂ pressure. This work shows the strong potential of polygenic analysis and targeted genetic modification for creation of cisgenic industrial brewer's yeast strains with specifically improved traits.