Primary mediastinal seminomas (MS) are rare tumors. Histologically, they are similar to their counterpart in the gonads. The survival rate has varied from 50% to 85% in different series. However, large series of these tumors primarily in the mediastinum are lacking. At the molecular level, a few reports document K-ras mutations in up to 40% of testicular seminomas (TS), localized predominantly to codon 12. Reports on TS p53 immunohistochemistry (IHC) range from negative to overexpression approaching 90% of cases, and by sequence analysis one small series showed a 23% mutation rate. To date, no analyses have been performed for either K-ras mutations or p53 immunohistochemical expression in primary MS. The authors studied 13 cases each of primary MS and TS from archival formalin-fixed, paraffin-embedded sections in which adequate tumor sampling and clinical history, including serological studies, and histological, histochemical, and IHC staining, were performed to confirm the diagnosis. p53 immunoperoxidase staining using citrate buffer/microwave antigen retrieval was performed. Topographic genotyping was performed on 5-microns-thick tissue sections up to 17 years old, in which the neoplastic cell population was sampled. Additionally, multiple sites within a given cases were sampled to determine clonality of the tumor cell population. Polymerase chain reaction and subsequent sequence analysis of the K-ras-2 exon-1 gene was used for mutation analysis. Focal weak staining with p53 IHC was observed in 4 of 13 (31%) MS and 10 of 13 (77%) TS cases, with all remaining cases being negative (P < .05). Only one MS case (8%) showed K-ras mutation (codon 13 GGC > GAC; glycine > aspartate), which is in contrast to 2 of the TS cases (15%), showing codon 12 mutations. All the remaining cases were wild type. Therefore, primary mediastinal seminomas appear to be different in their K-ras sequence and p53 immunostain profile from TS. Codon mutation type may be useful in determining primary versus metastatic origin of a mediastinal seminoma.