Fluorescence in situ hybridization （FISH， fluorescence in situ hybridization） is a powerful tool for molecular cytogenetics studies and is able to authentically allocate particular DNA or RNA sequences on chromosomes. With the development of the genome sequencing technology， the reduction of sequencing cost and the publishment of a large number of species genome information， Oligonucleotide （Oligo） probes based on high-throughput sequencing and reference genome were developed showing the advantages in FISH. In comparison with the traditional probes， Oligo-FISH can further reveal the evolution， inheritance and variation of chromosomes more precisely and deeply in plant evolution.This paper reviews the types and applications of target DNA and fluorescent probes in the development of fluorescence labeling， as well as the types and preparation techniques of oligonucleotide probes， mainly focusing on the origin and development of Oligo-FISH and its application in plants， which plays an important role in the identification of plant chromosomes and plant homologous chromosomes. Since the karyotype of species and genera can be constructed by Oligo-FISH technology， the results of Oligo-FISH can provide guidance for genome assembly of crops in this genus which have no complete genomes. Oligo painting can also solve the problem of fusion and exchange between chromosomes of heteropolyploid species and accurately detect whether there is translocation and heterologous recombination between chromosomes. Therefore， the development of Oligo-FISH technology provides strong support for the assembly of the genome at chromosome level. In the future， Oligo-FISH technology combined with signal amplification technology can overcome the challenge of low Oligo probes with high concentration of repeat sequences in regions， and visualize limited gene regions， such as the detection of promoters or enhancers or the localization of gene segments in transgenes. These studies will make better use of the research results of species genetics and evolution to further ensure， assist and innovate the improvement and development of crop genetics and breeding.