Isolates of the most important Puccinia species that have been reported on Chrysanthemum x morifolium were collected and the sequences of their ribosomal DNA internal transcribed spacers ITS1 and ITS2 were determined and used as phylogenetic markers. The focus of this study was on Puccinia horiana, due to its quarantine status and its impact in commercial chrysanthemum production. Three technical adjustments were needed to reliably obtain the nucleotide sequences starting from fresh or dried samples. The complete rDNA ITS nucleotide sequences of P. horiana, Puccinia chrysanthemi, and Puccinia tanaceti isolates of varying age and geographic origin were determined. We also identified an as yet undescribed Puccinia species on six old herbarium samples from chrysanthemum. This new species is morphologically similar to P. chrysanthemi and near identical to recent rust samples from Artemisia tridentata. P. tanaceti could not be confirmed as a pathogen of chrysanthemum. Different rDNA ITS sequences were present in P. horiana, with intra-isolate and inter-isolate variability in the length of three nucleotide repeat regions in the different rDNA tandem copies. We also identified three ITS types within P. horiana, with the rarer types displaying up to 67 bp nucleotide sequence differences. These rarer ITS types were detected at low copy number in all isolates. In general, very little rDNA ITS sequence variation was observed between P. horiana isolates from 1903 and 2003, and among isolates from different continents. Phylogenetic analyses using distance, Maximum Likelihood and Bayesian methods confirmed P. horiana, P. chrysanthemi, and the new Puccinia sp. as well-resolved groups, with P. horiana clustering in the clade where the economically important rust species of the Poaceae are located, and P. chrysanthemi and the new Puccinia sp. clustering in the clade where the majority of the rust fungi with hosts in the Asteraceae is located.