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Wheat is one of the most important cereals that supply needs of the human food in all countries. Fusarium head blight (FHB), caused primarily by Fusarium graminearum is one of the most destructive diseases of wheat that produces the mycotoxin deoxynivalenol (DON), a protein synthesis inhibitor which is harmful to human and livestock. Chemical modification in DON structure could reduce DON accumulation in the grain. AYT1 gene, encoding an acetyltransferase enzyme plays a major role in reducing the toxicity of DON. In this study, we used molecular techniques such as PCR, RT-PCR and southern blotting to confirm the insertion and proper expression of this transgene in the second generation of transgenic wheat plants. Obtained results showed the presence of Ayt1 gene in most of the evaluated transformed plants, and transgene expression was confirmed by RT-PCR. Moreover, southern blot analysis for evaluation of transgene copy number showed more than one copy in some of the transgenic lines.