Australia supports a high diversity of sequestrate (truffle-like) macrofungi. This has long been thought to be related to the predominantly or seasonally dry climate. The present study posits that if aridity were a key factor in the evolution of sequestrate fruit-bodies, most sequestrate species would have emerged in Australia only after it began to aridify, which occurred post-separation with Antarctica (c. 32 million years ago). Focusing on the high phylogenetic diversity of sequestrate taxa in the Agaricomycetes in Australia, dates of sequestrate nodes were compiled directly from published phylogenies (four lineages) or created using sequences available on GenBank that were processed in BEAST using a secondary calibration method (nine lineages). Although the morphologically diverse Hysterangiales was found to be the first group to become sequestrate, c. 83 million years ago, overall sequestration in Australia occurred more recently. Models were created and compared and support was found for an increased rate of sequestration in Australia at some point between 34 and 13 million years ago (during the Oligocene and Miocene). Although the rate of sequestration is shown to have increased in Australia after separation from Antarctica, the timing also overlaps with the radiation of potential mycorrhizal plant associates, and the emergence of specialised mycophagous marsupials. Although aridification is evidently not the sole driver of sequestration, it is still likely to have had a major influence on the diversity of sequestrate fungi in Australia. Comparisons with other regions of high sequestrate diversity will be informative.