In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand to localize a specific DNA or RNA sequence on a chromosome or section of tissue (in-situ) fixed on a slide. Flourescence in-situ hybridization (FISH) technique facilitates the localization of genes to different chromosomal locations. It is extensively applied as a gene mapping tool for identification and validation of cytogenetic aberrations identified through comparative genomic hybridization (CGH) on large cohort of archival samples in a tissue microarray format. The discovery of cytogenetic aberrations in cancer has led to the development of quite a few FDA approved molecularly targeted drugs for the management of patients undergoing cancer treatment. FISH technique is extensively utilized as a predictor of responsiveness to treatment with targeted inhibitors, residual disease monitoring and also in noninvasive methods for the detection of tumor cells. Furthermore detection of circulating tumor cells can be detected which have metastatic potential with poor survival prospects. With the development of high throughput technologies like comparative genomic hybridization CGH and next-generation DNA sequencing, human pathology archival specimens of human tumors in various stages of development, can be utilized in the post-human-genome-sequencing era to obtain diagnostic and therapeutic guidance. This article will discuss the extensive application of FISH in diagnosis, prognosis and therapeutic monitoring of cancer.