Genomic in situ hybridization (GISH) has been used to discriminate the different ancestral genome donors in polyploids or hybrids, analyze the phylogenetic relationships between cultivar and wild relatives, and study chromosome behavior of meiosis in plants.GISH technology includes multicolor genomic in situ hybridization (mcGISH), comparative genomic in situ hybridization (cGISH), and selfgenomic in situ hybridization (selfGISH). Its key procedures contain chromosome preparation, probe preparation and length optimization, the concentration ratio of probe to blocking DNA, and stringency of posthybridization washes. This paper reviewed the research progress on the development of GISH and its utilization in horticultural plants,and summarized their promising application prospects. During the past several years, the complete genome sequences are available for horticultural plants such as grape, banana, pear, sweet orange, and cucumber. It is necessary for researchers to select more chromosomal specific markers from the whole genomic sequence. Combing with fluorescence banding and fluorescence in situ hybridization (FISH) technologies, GISH will become a powerful tool to further illustrate the origin and identify genetic relationship of horticultural plants.