Recently, a proteasome β subunit expressed exclusively in thymic cortical epithelial cells was discovered in mice and humans. This subunit, designated β5t, is a component of the thymoproteasome, a specialized type of proteasome implicated in thymic positive selection. To investigate whether β5t could serve as a marker for the differential diagnosis of thymic epithelial tumors, we performed immunohistochemical analysis using anti-β5t antibody in 54 cases of thymic epithelial tumors comprising 41 cases of thymomas and 13 cases of thymic carcinomas. β5t was detected in the neoplastic epithelial cells of thymomas. Among the subtypes of thymoma, expression of β5t was observed in most cases of type B thymoma (20 of 21) but not in type A thymomas (0 of 3). In type AB thymomas, β5t expression was variable (6 of 17). Type B3 thymomas (4 cases) were positive for β5t but negative for CD5, c-kit, and glucose transporter 1 (GLUT-1), which are known as diagnostic markers for thymic carcinomas. In contrast, thymic carcinomas were negative for β5t (0 of 13) but expressed at least one and usually all of CD5, c-kit, and GLUT-1. Thus, β5t and CD5/c-kit/GLUT-1 were differentially expressed in type B3 thymoma and thymic carcinoma. We tested β5t expression in 39 cases of tumors arising from other organs, which showed the specific expression of β5t in thymic epithelial tumors. This study demonstrates that β5t is expressed in most type B and in some type AB thymomas and is a marker useful in differentiating type B3 thymomas from thymic carcinomas when used in combination with other diagnostic markers.