Fossils from the Eocene of South Australia and Western Australia and the Oligo–Miocene of Victoria represent the first known Australian leaf fossils of subfamily Persoonioideae, tribe Persoonieae. Persoonieaephyllum blackburnii sp. nov. is described from Middle Eocene Nelly Creek sediments near Lake Eyre, South Australia. Persoonieae are an important clade for understanding vegetation transitions in Australasia. The Nelly Creek leaf fossils are small (∼6 mm wide) and belong to an assemblage that has some characteristics of open vegetation, which is also inferred for the Oligo–Miocene of the Latrobe Valley, Victoria. In contrast, the Western Australian Late Eocene Persoonieae occur with diverse Lauraceae and other elements now typical of closed rainforests, and may, therefore, have been derived from communities that are unlike those in which most Persoonieae now occur. All fossil Persoonieae leaves so far known are hypostomatic (or virtually so), a state of stomatal distribution now only found in species of reasonably mesic habitats in New Zealand, New Caledonia and eastern Australian eucalypt forests. The ancestral state of stomatal distribution in Persoonieae leaves is unclear, but evidence suggests ancient associations of amphistomaty with open habitats, evolutionary loss of adaxial stomata in more closed vegetation, and the evolution of pronounced xerophylly within south-western Australian heathlands.