Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH.Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid-tolerant yeast as a metabolic engineering host, sevenZ. bailiistrains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. TheZ. bailiiNRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN-ARS-based CRISPR-Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion ofADE2and site-specific integration ofRhizopus oryzae ldhAcoding for lactate dehydrogenase into thePDC1locus. The resultingpdc1::ldhAstrain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR-Cas9 system inZ. bailii, which can be applied for a fundamental study of the species.