Fluorescence In Situ Hybridization (FISH) is a powerful molecular cytogenetic technique utilized for the visualization and localization of specific DNA sequences within chromosomes and interphase chromatin. This method has evolved significantly since its inception in the 1960s, with the introduction of non-radioactive probes that have enhanced safety and efficacy. The technique employs fluorescently labeled nucleic acid probes that, after denaturation, anneal to complementary target DNA at lower temperatures. Subsequent washing steps ensure the removal of non-specifically bound probes, allowing precise visualization of hybridization signals via fluorescence microscopy. FISH offers a versatile approach for detecting various genomic features, including centromeres, telomeres, gene loci, and chromosomal rearrangements. The success of FISH largely depends on the choice of probes, which can be categorized into traditional and advanced types. Traditional probes, such as Bacterial Artificial Chromosomes (BAC) and ribosomal DNA, are larger and cloned using diverse vectors, while advanced probes consist of short synthetic oligonucleotides that provide enhanced sensitivity and specificity. This technique has broadened the field of cytogenetics, facilitating the detailed study of chromosomal structure and function in both plant and animal sciences. Its applications span genome mapping, evolutionary studies, clinical diagnostics, and the investigation of chromosomal abnormalities, making FISH a cornerstone of modern molecular cytogenetics. This chapter provides a comprehensive overview of FISH, detailing its principles, probe types, and significant applications, reflecting its central role in advancing our understanding of genomic organization and dynamics.