Fluorescence in situ hybridization (FISH) is a powerful, morphology-based technique to assess targeted copy number alterations or gene rearrangements in formalin-fixed, paraffin-embedded tissues. It has a wide range of applications in routine clinical contexts to identify cytogenetic biomarkers for more accurate diagnosis and prognostic stratification. This review and update addresses practical uses of FISH as a molecular diagnostic tool in the setting of brain tumors, including gliomas, embryonal neoplasms, ependymomas and meningiomas, focusing on key genetic biomarkers, such as 1p19q codeletion, epidermal growth factor receptor (EGFR) gene amplification, BRAF rearrangement and many others. Also discussed are lessons learned over the past decade, including common technical issues to consider when implementing and interpreting FISH results in a clinical setting. © 2010 International Society of Neuropathology.