Sequences of the 5S rDNA repeat have been determined from four outgroup taxa (Arillastrum group), 2 Angophora species, and 19 Eucalyptus species (including representatives from all informal subgenera). Alignment shows that, as in other plants, the tandemly repeated 5S rRNA genes are highly conserved while the noncoding intergenic spacers are variable. A 50-base pair (bp) repeating element, which has undergone duplication and modification in certain taxa, is identifiable within the spacer and accounts for much of the variability. There were several other multiresidue deletion/insertion events in the spacers that define major clades. Based on the indel events and modifications of the 50-bp element it is apparent that the spacer from bloodwood (informal subgenera Blakella and Corymbia) species of Eucalyptus is more similar to that of Angophora than to non-bloodwood species of Eucalyptus. Parsimony analysis showed Eucalyptus to be paraphyletic, with the bloodwoods being more closely related to Angophora than to the rest of Eucalyptus. This finding supports taxonomic revision of Eucalyptus.