Lager brewing yeasts (Saccharomyces pastorianus), the natural hybrids of S. cerevisiae and S. eubayanus, are usually heterothallic polyploidy or aneuploidy. Their intricate ploidy is a great challenge to genetic studies and strain improvement. Haploid breeding is an effective method to overcome these difficulties. Also, haploid strains play an important role in scientific research and breeding. However, lager brewing yeasts only divide asexually and hardly bear spores under normal conditions, so it is very difficult to get haploid strains from them. In this study, we established comprehensive methods to induce, separate and identify haploid strains of industrial brewer's yeast.First, we selected efficient sporulation medium to induce the sporulation of an industrial brewer's yeast strain G-03, and ther isolated spores from vegetative cells and formed colonies on YPD plates. After that, flow cytometry was used to determine the ploidy types of the pre-judged haploid candidates. Ultimately, we analyzed the genotypes of the segregants by PCR reaction and mating test in order to get precise results.Using this protocol, we obtained 26 yeast segregants by spore isolation, and 4 of them pre-judged as haploid candidates were finally confirmed as haploid by flow cytometric analysis. Two of them were MATa and others were MATα. By scanning electron microscope (SEM), the cells of 4 haploid segregants showed similar morphology to each other but had obvious differences compared with the parent strain. Pseudohyphal growth occurred in parent cells after long-period cultivation but none was found in haploid segregants.Sporulation of industrial brewer's yeast and germination of their spores was difficult but not impossible. Nevertheless, the screening and identification of haploid segregants were more challenging.