During the past two decades fluorescent in-situ hybridization (FISH) has become a standard technique to directly localize, orient, and order genes in the genomes of a wide range of species. Despite the availability of a variety of probes, probe labeling and signal-detection systems, and advanced image analysis software, the core procedures used to carry out FISH remain the same. A detailed overview of these procedures, including target preparation (metaphase/interphase chromosomes and DNA fibers), probe labeling, in-situ hybridization, signal detection, and imaging, is here provided in a stepwise manner.