To determine the origin of the tetraploid annuals Microseris campestris and M. acuminata, chloroplast RFLP, RAPD and ITS sequence variability among nine populations of the two polyploids and 14 populations of the diploid annuals M. elegans and M. douglasii have been surveyed. Previously described variable chloroplast restriction sites infer M. douglasii as the possible maternal parent of both tetraploid species. However, the chloroplast genome typical for M. douglasii has now also been found in some plants of M. elegans. RAPD analysis revealed 172 polymorphic DNA markers that defined all four species as genetically distinct groups, but demonstrated closer associations between M. douglasii and M. acuminata, and between M. elegans and M. campestris. Sequencing of the ITS-1 and ITS-2 region yielded 73 phylogenetically informative sites. Thirty base-pair mutations separated the annual Microseris species from the outgroup, Uropappus lindleyi. The putative interspecific allotetraploid M. campestris contained only one type ITS sequence that, on the basis of eight synapomorphic substitutions was derived from M. elegans. The single ITS of M. acuminata shares six common sites with M. douglasii. Surprisingly, six sites were synapomorphic for the two tetraploids, M. campestris and M. acuminata, suggesting recombination within the ITS of both species with that of a common, now extinct, parental taxon, possibly the donor of the M. douglasii type chloroplasts found in both tetraploids. These results confirm the interpretation of M. campestris as derived from M. douglasii (extinct population) and M. elegans, and resolve the unknown origin of M. acuminata as an intraspecific hybrid between two very distinct populations of M. douglasii, one of them the same extinct M. douglasii form that contributes to M. campestris.