Thymic squamous cell carcinoma (TSQCC), accounting for 70-80% of thymic carcinoma cases, is distinct from thymoma. However, differential diagnosis for type B3 thymoma is sometimes challenging, even with established markers for TSQCC, including KIT and CD5, which are expressed in ~ 80% of TSQCCs and ~ 3% of thymomas. Novel TSQCC-specific markers would facilitate precise diagnosis and optimal treatment. Herein, we found that preferentially expressed antigen in melanoma (PRAME) may be a novel TSQCC-specific diagnostic marker. We comprehensively profiled 770 immune-related mRNAs in 10 patients with TSQCC and two healthy controls, showing that PRAME and KIT were significantly upregulated in TSQCC (adjusted p values = 0.045 and 0.0011, respectively). We then examined PRAME expression in 17 TSQCCs and 116 thymomas via immunohistochemistry. All 17 (100%) TSQCCs displayed diffuse and strong PRAME expression, whereas eight of 116 (6.8%) thymomas displayed focal and weak expression (p < 0.0001). KIT and CD5 were positive in 17 (100%) and 16 (94.1%) TSQCCs, respectively, whereas one (0.9%) type B3 thymoma showed double positivity for KIT and CD5. The KIT-/CD5-positive type B3 thymoma was negative for PRAME. Thus, combinatorial evaluation of PRAME with KIT and CD5 may facilitate a more precise diagnosis of TSQCC.