Multi‐color (or multi‐marker) fluorescence in situ hybridization (mFISH) is a well‐established, valuable, complementary tool for prenatal and pathological (tumor) diagnosis. A variety of chromosomal abnormalities, such as partial or total chromosomal gains, losses, inversions, or translocations, which are considered to cause genetic syndromes, can relatively easily be detected on a cell‐by‐cell basis. Individual cells either in suspension (e.g., in the form of a cytological specimen derived from body fluids) or within a tissue (e.g., a solid tumor specimen or biopsy) can be quantitatively evaluated with respect to the chromosomal hybridization markers of interest (e.g., a gene or centromeric region) and with due consideration of cellular heterogeneity. FISH is helpful or even essential for the (sub‐)classification, stratification, and unambiguous diagnosis of a number of malignant diseases and contributes to treatment decision in many cases. Here, the diagnostic power and limitations of typical FISH and mFISH approaches (except chromosome painting and RNA hybridization) are discussed, with special emphasis on tumor and single‐cell diagnostics. Well‐established and novel FISH protocols, the latter addressed to accelerate and flexibilize the preparation and hybridization of formalin‐fixed and paraffin‐embedded tissues, are provided. Moreover, guidelines and molecular aspects important for data interpretation are discussed. Finally, sophisticated multiplexed approaches and those that analyze very rare single‐cell events, which are not yet implemented in diagnostic procedures, will be touched upon. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.This article was corrected on 21 December 2023. See the end of the full text for details.Basic Protocol 1: (m)FISH applied to formaldehyde‐fixed paraffin‐embedded tissuesBasic Protocol 2: (m)FISH applied to cytological specimens