Claudins are tight junction proteins with claudin-6 (CLDN6) expression mostly restricted to embryonic and fetal life. Previously reported gene expression microarray analysis showed an increased level of CLDN6 in atypical teratoid rhabdoid tumors (AT/RT) compared with other central nervous system (CNS) tumors and sarcomas. However, there exist conflicting data on expression of CLDN6 as assessed by immunohistochemistry in CNS tumors. We established membranous staining as a specific and reproducible method for evaluating CLDN6 expression based on fetal and adolescent controls. We then evaluated a large group (257) of pediatric tumors using tissue microarrays, including: 47 malignant rhabdoid tumors (MRTs), (31 AT/RTs and 16 non-CNS MRTs); 67 small, round, blue cell tumors (10 Wilms tumors, 10 embryonal rhabdomyosarcomas, 10 neuroblastomas (NBs), 10 synovial sarcomas (SSs), 9 hepatoblastomas (HBs), 9 alveolar rhabdomyosarcomas, and 9 Ewings sarcomas); and 143 CNS tumors (24 medulloblastomas, 21 pilocytic astrocytomas, 14 astrocytomas grade II/III, 13 gangliogliomas, 12 glioblastomas, 12 ependymal tumors, 11 choroid plexus tumors, 10 meningiomas, 8 dysembryoplastic neuroepithelial tumors, 8 oligodendrogliomas, 4 craniopharyngiomas, 2 germinomas, 2 primitive neuroectodermal tumors (PNET), and 2 central neurocytomas). CLDN6 expression was seen in 12 of 31 (39%) AT/RTs, 7 of 16 (44%) non-CNS MRTs, 5 of 10 (50%) Wilms tumors, 1 of 9 (11%) HBs, 2 of 2 (100%) germinomas, 1 of 2 (50%) CNS PNETs, 1 of 24 (4%) medulloblastomas, and 1 of 10 (10%) meningiomas. Ten of 11 (91%) choroid plexus tumors showed apical staining but no concentric membranous staining. Although CLDN6 is expressed in both AT/RTs and MRTs, it is not a specific biomarker as it is expressed in a variety of other pediatric CNS and soft tissue tumors.