A variety of multicolor fluorescence in situ hybridization (FISH) assays have been developed in the last decade. Routine application of such techniques started in 1996 with the simultaneous use of all the 24 human whole chromosome painting probes (i.e. multiplex-FISH = M-FISH and spectral karyotyping = SKY). Since that time different approaches for chromosomal differentiation based on multicolor-FISH (mFISH) assays have been published with the purpose to characterize structurally abnormal chromosomes and supernumerary marker chromosomes of unknown origin after conventional karyotypic analysis. Their characterization is of high clinical impact and is the requisite condition for further molecular investigations aimed at identification of disease related genes. We present an overview of the available different mFISH methods, highlighting their advantages and limitations, as well as their applications in clinical and tumor cytogenetics. Finally, an outlook is given on further possible developments of thi s special field of molecular cytogenetics.