Diuris is a terrestrial orchid genus of at least 61 and possibly more than 100 species, restricted to Australia except for one species endemic in Timor. Distinctive species groups have respective eastern and western centres of distribution. Although species affinities have been vaguely understood for many years, no formal infrageneric treatment has been undertaken as Diuris possesses few reliable morphological characters for a classification system. We have undertaken cladistic parsimony and Bayesian phylogenetic analyses of Diuris by using the ITS1–5.8S–ITS2 region of nuclear rDNA and morphological characters, with a subset of samples also studied by amplified fragment length polymorphism (AFLP) as an independent test of phylogenetic relationships. Four major clades with strong bootstrap support were resolved and are named here according to a recently published classification; D. sulphurea forms a lineage (subg. Paradiuris) of its own that is well supported as the sister to the rest of Diuris. Two other major eastern clades contained species related to D. maculata (subg. Xanthodiuris) and D. punctata (subg. Diuris), respectively. Although these latter two subgenera are genetically well resolved, there is minimal genetic variation at species level, consistent with recent, rapid speciation. A fourth clade (subg. Hesperodiuris) has a centre of distribution in Western Australia, and has more genetic and morphological variation than the eastern subgenera. Total evidence analysis provides support for the western clade being sister group to the two eastern subgenera Diuris and Xanthodiuris; however, this relationship was not resolved by molecular data. Hybridisation is known to be common among species within subgenera Diuris and Xanthodiuris. Instances of incongruence between different datasets were found suggestive of hybridisation events between species of different sections of Diuris.