Bird cytogenetics has advanced significantly in recent decades, largely due to the application of fluorescence in situ hybridization (FISH), a powerful technique for mapping specific DNA sequences on chromosomes. Most bird species retain a karyotype similar to the putative ancestral avian karyotype, with a diploid number (2n) of approximately 80, consisting of a few pairs of macrochromosomes and numerous microchromosomes. Despite this overall karyotypic conservation, various species exhibit extensive chromosomal rearrangements, making cytogenetic studies essential for understanding avian genome organization and evolution. FISH has become a fundamental tool in avian cytogenetics, enabling precise identification of chromosomal rearrangements, gene mapping, and comparative analyses of chromosomal synteny across species. The technique involves the isolation and labeling of specific DNA probes, followed by their hybridization onto metaphase chromosomes to visualize genomic regions of interest. Advances in probe development, including whole-chromosome paints, bacterial artificial chromosome (BAC) probes, and repetitive sequence markers, have significantly enhanced the resolution of avian karyotypic studies. This chapter highlights the methodologies currently employed in avian FISH, encompassing probe preparation, hybridization protocols, and data interpretation.