Subgenus Tridentatae (Artemisia, Asteraceae) can be considered a polyploid complex. Both polyploidy and hybridization have been documented in the Tridentatae. Fluorescent in situ hybridization (FISH) and fluorochrome banding were used to detect and analyze ribosomal DNA changes linked to polyploidization in this group by studying four diploid-polyploid species pairs. In addition, genome sizes and heterochromatin patterns were compared between these populations. The linked 5S and 35S rRNA genes are confirmed as characteristic for Artemisia, and a pattern at the diploid level of three rDNA loci located at telomeric positions proved to be typical. Loss of rDNA loci was observed in some polyploids, whereas others showed additivity with respect to their diploid relatives. Genome downsizing was observed in all polyploids. Banding patterns differed depending on the pair of species analysed, but some polyploid populations showed an increased number of heterochromatic bands. FISH and fluorochrome banding were useful in determining the systematic position of Artemisia bigelovii, for which a differential pattern was found as compared with the rest of the group. Additionally, FISH was used to detect the presence of the Arabidopsis-type telomere repeat for the first time in Artemisia.