Fluorescent in situ hybridization (FISH) represents a moden molecular pathology technique, alternative to conventional cytogenetics (karyotyping). In addition to metaphase spreads, it can be applied directly to interphase nuclei. The latter makes the FISH technique powerful for pathologists for it integrates molecular genetics and classic cytogenetics and brings them together to a single framework for morphologic evaluation. Interphase FISH can be applied to imprints from fresh tissue or to paraffin sections after proteinase K digestion. Centromeric, telomeric and locus DNA-sequence specific probes can be used to identify aneuploidy or gene mutations. Several protocols combine molecular cytogenetics with classic karyotyping. Other sophisticated, FISH-based protocols have been introduced. Among them, comparative genomic hybridization is very important for it can detect non-balanced chromosomal aberrations of uncultured tumor cells and provide overall genomic information in a single experiment. This review presents the principles and applications of FISH technique for the investigation of the cytogenetic background of pituitary adenomas.