Thymomas are rare mediastinal tumors exhibiting heterogeneous behavior. Although histologic subtypes and stages serve as prognostic factors, the molecular mechanisms of thymoma progression are unclear. Immunohistochemical markers like Bcl-2, D2-40, β-catenin, and E-cadherin offer insights into thymoma biology, but their predictive value for clinical outcomes remains uncertain. This study evaluated the expression of these markers across thymoma subtypes and stages, aiming to assess their prognostic significance. A retrospective analysis was conducted on 66 thymoma cases resected at a single center between 2005 and 2023. Immunohistochemical staining was performed to assess the expression of Bcl-2, D2-40, β-catenin, and E-cadherin. Clinicopathological characteristics were correlated with immunohistochemical findings using statistical analysis. Differential expression patterns of Bcl-2, D2-40, β-catenin, and E-cadherin were observed across thymoma subtypes and clinical stages. Bcl-2 displayed cytoplasmic positivity predominantly in type A and B thymomas, while E-cadherin showed membranous staining in type B thymomas and cytoplasmic staining in type A and AB thymomas. β-catenin demonstrated membranous staining in type B thymomas and cytoplasmic staining in type A and AB thymomas. D2-40 expression was localized to peripheral regions and invasive nests of thymomas, with higher expression in type B2 thymomas and early-stage tumors. Our findings indicate that immunohistochemical markers may provide valuable insights into thymoma biology and prognosis. Further validation in larger, multicenter cohorts is warranted to confirm the prognostic significance of these markers and their potential utility in guiding clinical management.