Fluorescence in situ hybridization(FISH) , characterized to be fast,sensitive,accurate and effective, is a new technique of in situ hybridization which can directly locate specific DNA or RNA sequence on chromosomes by using biotin labelling probes. This article summarized the application of fluorescence in situ hybridization in crop genetics and breeding researches,including: 1)screening repetitive DNA sequences and multicopy gene families; 2)identifing the alien chromosomes and chromosome segments in allopolyploids; 3)detecting and locating low or single copy DNA sequence. With some new techniques developed, the FISH method will play important roles in much more fields of basic and applied researches in crop genetics and breeding.