Molecular cytogenetics includes a spectrum of methodologies that use molecular reagents to better define chromosomal alterations in normal and neoplastic cells. Brain tumors are a group of neoplasms for which there is a wealth of cytogenetic and molecular genetic information, and some of the newer techniques have extended the types of samples from which genetic information which can be obtained to biopsies and even paraffin-embedded sections. Fluorescence in situ hybridization on interphase nuclei has been used to confirm gains of chromosome 7, loss of chromosome 10, 9p deletion and gene amplification in malignant gliomas, and to visualize isochromosome 17q in medulloblastomas. Comparative genomic hybridization uses genomic DNA to determine gains and losses of chromosomes and chromosomal regions. This approach is particularly useful for identifying gene amplification. For cases in which chromosomal spreads are obtained, chromosomal painting is helpful in determining the origin of chromosomal segments. Several methods are now available in which each of the 22 autosomes and the sex chromosome can be identified by unique colors, termed Spectral karyotyping and multiplex-FISH. These molecular cytogenetic techniques are important clinical and experimental tools that have provided new insight into the genetic alterations of brain tumors.