In situ hybridization involves hybridization of DNA or RNA probes to the cytological preparations. The technique originally used auto-radiographic labeling to map both repetitive and low copy DNA sequences. The problem associated with this technique was its short half life, lack of safety and long exposure time which hindered its widespread use in DNA hybridization. To overcome these problems, non isotopic in situ hybridization was developed for use in animal and plant species. In the last decade, the development of haptens and fluorochromes enabled simultaneous multicolored detection of differentially labeled probes. Characterization of parental genomes in interspecific hybrids, restructured chromosomes, gene mapping, detecting nature of chromosome pairing, establishing phylogenetic relationship among the species and localizing introgressed segment have been successfully achieved by fluorescence in situ hybridization. Keywords: In situ hybridization, phylogenetic relationship, homoeologous pairin