Alterations of BRAF are the most common known genetic aberrations in pediatric gliomas. They frequently are found in pilocytic astrocytomas, where genomic duplications involving BRAF and the poorly characterized gene KIAA1549 create fusion proteins with constitutive B-Raf kinase activity. BRAF V600E point mutations are less common and generally occur in nonpilocytic tumors. The development of BRAF inhibitors as drugs has created an urgent need for robust clinical assays to identify activating lesions in BRAF. KIAA1549-BRAF fusion transcripts have been detected in frozen tissue, however, methods for FFPE tissue have not been reported. We developed a panel of FFPE-compatible quantitative RT-PCR assays for the most common KIAA1549-BRAF fusion transcripts. Application of these assays to a collection of 51 low-grade pediatric gliomas showed 97% sensitivity and 91% specificity compared with fluorescence in situ hybridization or array comparative genomic hybridization. In parallel, we assayed samples for the presence of the BRAF V600E mutation by PCR pyrosequencing. The data further support previous observations that these two alterations of the BRAF, KIAA1549 fusions and V600E point mutations, are associated primarily with pilocytic astrocytomas and nonpilocytic gliomas, respectively. These results show that fusion transcripts and mutations can be detected reliably in standard FFPE specimens and may be useful for incorporation into future studies of pediatric gliomas in basic science or clinical trials. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology.