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Ribosomal DNA (rDNA) is an excellent cytogenetic marker owing to its tandem arrangement and high copy numbers. However, comparative studies have focused more on the number of rDNA site variations within the Chrysanthemum genus, and studies on the types of rDNA sites with the same experimental procedures at the species levels are lacking. To further explore the number and types of rDNA site variations, we combined related data to draw ideograms of the rDNA sites of Chrysanthemum accessions using oligonucleotide fluorescence in situ hybridization (Oligo-FISH). Latent variations (such as polymorphisms of 45S rDNA sites and co-localized 5S-45S rDNA) also occurred among the investigated accessions. Meanwhile, a significant correlation was observed between the number of 5S rDNA sites and chromosome number. Additionally, the clumped and concentrated geographical distribution of different ploidy Chrysanthemum accessions may significantly promote the karyotype evolution. Based on the results above, we identified the formation mechanism of rDNA variations. Furthermore, these findings may provide a reliable method to examine the sites and number of rDNA variations among Chrysanthemum and its related accessions and allow researchers to further understand the evolutionary and phylogenetic relationships of the Chrysanthemum genus. [ABSTRACT FROM AUTHOR]