The incidence of retroperitoneal seminoma is much less than that of its gonadal counterpart. Accurate diagnosis of retroperitoneal seminoma is critical, because it carries an excellent prognosis due to its favorable response to radiation therapy and/or cisplatin-based chemotherapy. However, correctly diagnosing a retroperitoneal seminoma may be challenging, especially when the biopsy material is limited. The present study was conducted to evaluate histologic findings and immunohistochemical staining patterns in biopsy specimens of retroperitoneal seminoma and to compare their utility as diagnostic tools. Thirty biopsy specimens of retroperitoneal seminoma were assessed for histologic characteristics and immunohistochemical expression of OCT4, c-kit, placental-like alkaline phosphatase, and cytokeratin AE1/AE3. The clinical information, morphologic features, and staining intensities and the percentages of positively staining tumor cells were analyzed. The mean age of patients was 38 years. Lymphocytic infiltration and nucleolar prominence in tumor cells were found in all 30 cases (100%). The incidence of other histologic characteristics were as follows: fibrous septa/stroma in 80% (24 cases), clear tumor cell cytoplasm in 70% (21 cases), tumor necrosis in 60% (18 cases), cellular pleomorphism in 53% (16 cases), granulomatous inflammation in 50% (15 cases), distinct cell borders in 46% (14 cases), intercellular edema in 23% (7 cases), and syncytiotrophoblasts in 3% (1 case). The mean mitotic count was 3 (range 0 to 15) per 10 high-power fields. All 30 cases (100%) of retroperitoneal seminoma revealed moderate to strong nuclear OCT4 staining in more than 50% of tumor cells. Twenty-one cases (70%) showed membranous expression of c-kit by tumor cells, with moderate to strong staining intensity in most cases. Variable degrees of staining for placental-like alkaline phosphatase were identified in 23 cases (77%) with occasional background staining artifact. Six cases (20%) displayed a positive cytokeratin AE1/AE3 staining pattern with weak to moderate intensity. In conclusion, the most common histologic findings in limited biopsy specimens of retroperitoneal seminoma were lymphocytic infiltration and nucleolar prominence in tumor cell nuclei. OCT4 immunostaining, with its superior sensitivity and easy interpretation compared with other markers, is a powerful tool for confirming the diagnosis of retroperitoneal seminoma.