rDNA is abundant in various organisms, typically expressed as conserved tandem repeats. It plays a crucial role in ribosome synthesis, gene transcription, and expression, and it affects the occurrence of diseases in both animals and plants, aging, protein synthesis, genomic stability, and genome evolution across a wide range of organisms. Among the different types of rDNA, 35S rDNA (also referred to as 45S rDNA) and 5S rDNA are particularly important in plant research. The use of 35S rDNA and 5S rDNA as probes has enabled the study of chromosomal composition, revealing species characteristics that are valuable for crop breeding, evolutionary biology, systematics, and other fields. This review focuses on the application of 35S rDNA and 5S rDNA and discusses research findings on sugarcane and its related germplasm that have been obtained through fluorescence in situ hybridization. This information has provided a foundation for understanding the genetic relationships, genetics, breeding, and evolutionary classification of sugarcane.