For the global economy and public health, management of fungal diseases is essential. Aspergillus flavus causes danger for those who are afflicted through the production of aflatoxin, which affects both plants and animals. Chemical therapy that blocks the biosynthesis of ergosterol (Azoles) is widely used to manage A. flavus. However, the gradual development of fungicide resistance reduces efficacy and causes overuse or adverse effects. In the present study, 123 strains of A. flavus collected from various environments were used for fungicide (metconazole) susceptibility test, and mutations in the drug target genes (cyp51s) of all strains were investigated. In addition, population genome analysis of the selected 54 strains was conducted to investigate new resistance mechanisms. Based on the MIC value of 10 mgL-1, the 54 strains were divided into two groups, less-sensitive (28 strains) and more-sensitive (26 strains). Using the Tessel5 program, the difference between the two groups for all variants was statistically analyzed (P-value), and 12,469 exon-variants satisfying P<0.05 were identified among a total of 561,952 variants. Functional annotation was performed on exon-variants and genes with very diverse domains, such as the Major facilitator superfamily (MFS) domain and Transcription factor domain, were identified. Currently, a knock-out transformation method using the CRISPR-Cas system is being tested to confirm whether the function of the genes is related to drug resistance. Our study suggests the possibility of future medication development and effective fungus pathogen control.