Abstract: In situ hybridization is one of the most important techniques in plant molecular cytogenetics research. In this paper, we briefly introduced the origin and development of in situ hybridization, firstly. Then, application of genomic in situ hybridization (GISH) in plant cytogenetics research as well as fluorescence in situ hybridization (FISH) in plant physical mapping and chromosome identification were detailed reviewed. Some new techniques such as fiber-FISH, BAC-FISH, and immuno-FISH were also introduced in this paper. And a prospect about FISH was given out.