Fishes represent the most diversified group of extant vertebrates, and cytogenetic investigations have been undertaken in this group since the early 1900s. The development of the fluorescence in situ hybridization (FISH) technique enabled the transition from the “classical” (black-and-white) to the “molecular cytogenetic” (color) era. Over the last 30 years, all sorts of DNA repeats (including rDNAs, satellite DNAs, microsatellites, multigene families, and transposable elements) have been thoroughly mapped onto fish chromosomes, providing insights into karyotype, B-chromosomes’, and sex chromosomes’ evolution. This chapter provides a unified and revised protocol for FISH in fish chromosomes.