Ability to grow at low pH is a desirable feature for the yeast Saccharomyces cerevisiae in an industrial environment, because low pH, among many positive effects on cell metabolism, diminishes bacterial contamination. Acidotolerance is a polygenic trait, which means that there are multiple genes determining this trait. Therefore, such traits are relatively difficult to study. With the aim to obtain acidotolerant yeast strains we iteratively crossed 6 genetically different yeast strains. After each crossing we applied selection to the segregants and selected the most resistant segregant of the generation, the so-called winner. The winner of a generation was the segregant we used in the next crossing. The winner of the last generation was able to grow at lower pH then all the parental strains and all other generation winners. Thus, we have successfully improved acidotolarence yeast phenotype with iterative crossing and selection. For the identification of quantitative trait loci (QTLs), we crossed the winner of the last generation with the strain Y7092 and prepared an extremely large pool of segregants. The segregants in the extreme pool grew in selective medium with low pH, while the segregants in the average pool grew in a medium without selection. After sequencing of the pools’ DNA, we compared frequencies of single nucleotide variations between the extreme and the average pool using bioinformatics analysis. We predicted 45 potential QTLs and suggested 91 plausible causative genes for acidotolarance.