Primary mediastinal seminomas (MS) are rare tumors that are histologically similar to their testicular counterparts. Reports document KIT mutations in gastrointestinal stromal tumors, mastocytosis, and germ cell tumors. Although rare exon 17 mutations have been reported in gonadal seminomas, their mediastinal counterparts have not been studied. To determine whether primary MS harbor KIT mutations, eight formalin-fixed, paraffin-embedded primary MS were microdissected; KIT exons 11 and 17 were sequenced. Four (50%) of the eight cases demonstrated KIT exon 17 mutations. Two of the cases showed single monoallelic base pair alterations; one of these mutations were silent. The other two cases each demonstrated two monoallelic point mutations. In each case, one of these mutations results in protein sequence alteration, and the other is silent. Sequencing of cloned PCR products showed both the silent and amino acid-altering mutations to be found on the same allele (cis). The codon 816 mutation previously identified in mastocytosis and gonadal germ cell tumors was not observed. Non-neoplastic tissues from these patients did not demonstrate KIT mutations; exon 11 mutations were not seen in either tumors or normal tissues. Only the three cases in which amino acid-altering mutations were observed showed a predominantly cytoplasmic CD177 KIT immunohistochemical staining, whereas the one non-amino acid mutating and all wild-type cases were immunonegative. Our findings demonstrate a unique KIT sequence and expression pattern among MS. KIT sequencing may assist in differentiating primary from metastatic MS.