Candida glabrata (C. glabrata) is emerging as the second most common cause for invasive candidiasis and early studies indicate that it is relatively insensitive to azole treatment. Transcription factor Pdr1 is the key regulator of azole resistance genes in C. glabrata. In this study, a Flag sequence was introduced into amino terminus of pdr1 gene at its native locus in chromosome through homologous recombination in clinical isolates of C. glabrata. Western blotting analysis with anti-Flag antibody showed a single immunoreactive species matching the predicted size of Pdr1. Spot assay showed that mutants expressing Flag-Pdr1 were more resistant to fluconazole than wild-type. Real-time quantitative polymerase chain reaction showed that gene expression levels of cdr1 and pup1 in mutants expressing Flag-Pdr1 were significantly increased than those in wild-type strains.