Background: Hashimoto's thyroiditis (HT) is a T cell-mediated autoimmune disease that primarily affects females. IFN- γ is a critical cytokine that has been related to the pathogenesis of HT. Objectives: We aimed to evaluate serum and expression levels of interferon-gamma (IFN- γ) in Egyptian women with HT and to assess the association between serum and expression levels of IFN- γ with clinical and laboratory characteristics of HT. Methodology: This case-control study included 120 women with HT and 70 controls. IFN- γ mRNA expression was analyzed using real-time polymerase chain reaction. Serum IFN- γ was measured using enzyme-linked immunosorbent assay. Results: Serum IFN- γ level and the level of IFN- γ mRNA are both sensitive and specific to be used as diagnostic markers for HT with cut off values of 28.57 pg/ml and 3.55 respectively. Both showed a significant positive correlation with TPO-Ab and Tg-Ab, obesity indices, dyslipidemia, and TSH, while they have a negative correlation with FT3, FT4. Conclusions: Serum IFN- γ level and the level of IFN- γ mRNA are both sensitive and specific to be used as diagnostic markers for HT, significantly correlated with thyroid autoantibodies and thyroid function tests.