AbstractKlPdr1p is a single Kluyveromyces lactis homologue of Saccharomyces cerevisiae ScPdr1p/ScPdr3p, the main transcriptional regulators of genes involved in S. cerevisiae multidrug resistance. KlPDR1 deletion leads to a sharp increase in K. lactis drug susceptibility. The presence of putative PDRE and YRE regulatory elements in the KlPDR1 gene promoter suggests an autoregulation of its transcription as well as its control by KlYap1p, the transcription factor involved in oxidative stress response. In this study, one plasmid‐borne Klpdr1‐1 allele that led to amino acid substitution (L273P) in the KlPdr1p was isolated. Overexpression of the Klpdr1‐1 allele from a multicopy plasmid in the K. lactis wild‐type and Klpdr1Δ mutant strain increased the tolerance of transformants to oligomycin. The plasmid‐borne Klpdr1‐1 allele increased the activation of the ScPDR5 promoter and complemented the drug hypersensitivity of the S. cerevisiae pdr1Δ pdr3Δ mutant strain. The results indicate that L273P amino acid substitution is the result of a gain‐of‐function mutation in the KlPDR1 gene that confers KlPdr1p hyperactivity, as revealed by a high expression of the ABC transporter gene KlPDR5, leading to multidrug resistance and rhodamine 6G efflux out of the cells. Copyright © 2013 John Wiley & Sons, Ltd.