The technique of in situ hybridization (ISH) using radioactively labeled DNA probes was first described in the late 1960s and early 1970s. The first use of fluorescence in situ hybridization (FISH) was reported in 1980s where RNA labeled with a fluorophore at the 3' end was used to detect specific DNA sequences. Since then, the technique has undergone various modifications for detecting single genes, chromosomes and whole genomes on various targets such as interphase nucleus, prematurely condensed chromosomes, metaphase chromosomes, fresh and paraffinized tissue sections. Although FISH is quite frequently used in clinical diagnostics, its use has recently been extended to the field of radiation biodosimetry where both stable (translocations) and unstable aberrations (dicentrics and rings) are detected with high resolution for estimating the absorbed radiation dose in humans after incidental, accidental or occupational exposure to ionizing radiation. This review summarizes the diverse applications of FISH in radiation biodosimetry that range from radiation dose estimation to prediction of deterministic (i.e. acute radiation syndrome severity) and stochastic (i.e. genetic mutations and cancer) effects in the affected human population.