This study aimed to investigate azole resistance mechanisms in Aspergillus flavus, which involve cyp 51A and cyp 51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of cyp 51A and cyp 51B genes for 34 A. flavus isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of cyp 51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in cyp 51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in cyp 51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the cyp 51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the cyp 51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the cyp 51A and cyp 51B genes is the primary mechanism responsible for resistance in A. flavus collection. Nevertheless, other resistance mechanisms can be involved.