BACKGROUND Fluorescence in situ hybridization (FISH) assays are indispensable in diagnostics and research. Routine application of this so-called molecular cytogenetic technique on human chromosomes started in 1986. Since then, a huge variety of different approaches for chromosomal differentiation based on FISH has been described. It was established to characterize marker chromosomes identified in conventional banding analysis as well as cryptic rearrangements not resolved by standard cytogenetics. OBJECTIVE/METHOD Even though molecular cytogenetics, like banding cytogenetics for almost 40 years, is often called dead now, it offers unique possibilities of single cell analysis. Thus, a review is presented here on the available diagnostic-relevant FISH methods and probe sets applied in routine pre- and postnatal clinical as well as tumor cytogenetics. CONCLUSION Molecular cytogenetics is a fast, straightforward and reliable tool that is indispensable in cytogenetic diagnostics. It is and will continue to be of high clinical impact in diagnostics, especially in the overwhelming majority of routine cytogenetic laboratories that cannot afford and do not need high-throughput chip-based platforms for their daily work.