Separation of pilocytic astrocytoma from diffuse astrocytomas frequently poses problems mostly related to small sample size. Precise classification and grading are essential due to different therapeutic strategies prompted by diagnoses of pilocytic astrocytoma WHO grade I, diffuse astrocytomas WHO grade II or anaplastic astrocytoma WHO grade III. Recently, genomic aberrations with a high specificity for distinct glioma entities have been described. Pilocytic astrocytomas carry a duplication at chromosome band 7q34 containing a BRAF-KIAA1549 gene fusion in the majority of cases. IDH1 mutations are observed very frequently in adult astrocytomas and IDH2 mutations have been reported in some astrocytomas. We examined a series of 120 astrocytomas including 70 pilocytic astrocytomas WHO grade I and 50 diffuse astrocytomas WHO grade II for both, BRAF-KIAA1549 fusion with a newly developed FISH assay and mutations in IDH1 and IDH2 by direct sequencing. Pilocytic astrocytomas contained the BRAF fusion in 49 cases (70%) but neither IDH1 nor IDH2 mutations. Astrocytomas WHO grade II exhibited IDH1 mutations in 38 cases (76%) but neither IDH2 mutations nor BRAF fusions. Thus, combined molecular analysis of BRAF and IDH1 is a sensitive and highly specific approach to separate pilocytic astrocytoma from diffuse astrocytoma.