Aspergillus flavus has been frequently reported as the leading cause of invasive aspergillosis in certain tropical and sub-tropical countries.Two hundred A. flavus strains originating from clinical and environmental sources and collected between 2008 and 2015 were phylogenetically identified at the species level by analyzing partial β-tubulin and calmodulin genes. In vitro antifungal susceptibility testing was performed against antifungals using the EUCAST (European Committee on Antimicrobial Susceptibility Testing) broth microdilution method. In addition, genotyping was performed using a short tandem repeat assay of a panel of six microsatellite markers (STR A. flavus 2A, 2B, 2C, 3A, 3B, and 3C), in order to determine the genetic variation and the potential relationship between clinical and environmental isolates.The geometric means of the minimum inhibitory/effective concentrations (MICs/MECs) of the antifungals across all isolates were (in increasing order): posaconazole, 0.13 mg/L; anidulafungin, 0.16 mg/L; itraconazole, 0.29 mg/L; caspofungin, 0.42 mg/L; voriconazole, 0.64 mg/L; isavuconazole, 1.10 mg/L; amphotericin B, 3.35 mg/L; and flucytosine, 62.97 mg/L. All of the clinical isolates were genetically different. However, identical microsatellite genotype was found between a clinical isolates and two environmental strains.In conclusion, posaconazole and anidulafungin showed the greatest in vitro activity among systemic azoles and echinocandins, respectively. However, majority of A. flavus isolates showed reduced susceptibility to amphotericin B. Antifungal susceptibility of A. flavus was not linked with the clinical or environmental source of isolation. Microsatellite genotyping may suggest an association between clinical and environmental strains, although this requires further investigation.