Currently, the molecular mechanisms of azole resistance in C. glabrata are unresolved. This study aims to detect azole resistance of C. glabrata after exposure to fluconazole (Diflucan) in vitro. After 50 days of induction, the five susceptible isolates of C. glabrata demonstrated cross-resistance to azoles (fluconazole (Diflucan), voriconazole and itraconazole). Mutations in PDR1 or ERG11 genes are key nodes in azole resistance of C. glabrata. DNA-Sequencing revealed three(3/5) fluconazole (Diflucan)-resistant isolates had undergone missense mutations (R376Q, R772K, E1083K in PDR1 and F135L in ERG11), all of which were newly discovered and previously unreported. mRNA expression of resistant genes in five resistant isolated was elevated, with CDR1 being the most prominent. Analysis using flow cytometry revealed that resistant strains showed decreased R6G uptake and increased efflux efficiency, but no obvious significance difference in biofilm production. C. glabrata acquires azole cross-resistance upon continuous exposed to fluconazole (Diflucan) and could remain resistant without antifungal agents. The development of azole resistance in C. glabrata has been linked to genes associated with efflux pump transporters and the ergosterol synthesis pathway. However, the relationship between resistance and newly discovered missense mutation sites requires further investigation.