Nucleic acid hybridization techniques have contributed significantly to the understanding of gene organization, regulation and expression. In situ hybridization is a method for detecting specific nucleotide sequences by using a labeled complementary nucleic acid probe. The power of in situ hybridization can be greatly extended by the simultaneous use of multiple fluorescent colors. Multicolor fluorescence in situ hybridization (FISH), in its simplest form, can be used to identify as many labeled features as there are different fluorophores used in the hybridization. Key methodological advances have allowed facile preparation of low-noise hybridization probes, and technological breakthroughs now permit multi-target visualization and quantitative analysis – both factors that have made FISH accessible to all and applicable to any investigation of nucleic acids. In the future, this technique is likely to have significant further impact on live-cell imaging and on medical diagnostics.