Talaromyces marneffei (T. marneffei) is a life-threatening dimorphic fungus endemic to southern China and Southeast Asia, with triazole resistance emerging as a critical clinical challenge in immunocompromised populations, particularly HIV/AIDS patients. This study aimed to characterize the molecular mechanisms underlying triazole resistance in clinical T. marneffei isolates from HIV/AIDS patients in China. Antifungal susceptibility testing was performed via broth microdilution according to CLSI M27-A4 guidelines. Whole-genome sequencing (WGS) and Sanger sequencing were used to identify genetic mutations associated with resistance, with a focus on ergosterol biosynthesis genes Erg3 and cyp51B. Sequence conservation analysis was conducted for candidate mutation sites. All four isolates exhibited cross-resistance to fluconazole and voriconazole, with markedly elevated minimum inhibitory concentrations (MICs) compared to the wild-type strain. WGS and sanger sequencing identified a novel heterozygous Erg3 c.1195G > A substitution (p.T208I) in three resistant isolates. The T208 residue is highly conserved in T. marneffei and other pathogenic fungi. This is the first report of an Erg3 T208I substitution linked to cross-resistance to fluconazole and voriconazole in T. marneffei, expanding the known genetic mechanisms of antifungal resistance in this pathogen.