AbstractThe budding yeast, Saccharomyces cerevisiae, has been widely used for genetic studies of fundamental cellular functions. The isolation and analysis of yeast mutants is a commonly used and powerful technique to identify the genes that are involved in a process of interest. Furthermore, natural genetic variation among wild yeast strains has been studied for analysis of polygenic traits by quantitative trait loci mapping. Whole‐genome sequencing, often combined with bulk segregant analysis, is a powerful technique that helps determine the identity of mutations causing a phenotype. Here, we describe protocols for the construction of libraries for S. cerevisiae whole‐genome sequencing. We also present a bioinformatic pipeline to determine the genetic variants in a yeast strain using whole‐genome sequencing data. This pipeline can also be used for analyzing Schizosaccharomyces pombe mutants. © 2019 by John Wiley & Sons, Inc.Basic Protocol 1: Generation of haploid spores for bulk segregant analysisBasic Protocol 2: Extraction of genomic DNA from yeast cellsBasic Protocol 3: Shearing of genomic DNA for library preparationBasic Protocol 4: Construction and amplification of DNA librariesSupport Protocol 1: Annealing oligonucleotides for forming Y‐adaptersSupport Protocol 2: Size selection and cleanup using SPRI beadsBasic Protocol 5: Identification of genomic variants from sequencing data