The F-35 AU-001 and AU-002 departed the Avalon Air Show in Geelong a day late due to poor weather at Amberley.
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The aircraft were scheduled to leave the air show to fly to RAAF Base Amberley in Queensland on Sunday, before returning to the US for training, but departed Monday instead.
The aircraft requires modifications for lightning protection and these modifications have not yet been completed on the visiting Australian aircraft.
While the visiting aircraft belong to Australia, they are currently placed within a training pool in the US and the specific movements of the aircraft are authorised by the US.
The aircraft will be modified with lightning protection before they return to Australia next.
The aircraft touched down in Australia for the first time ever at the Avalon International Airshow on Friday.
Prime Minister Malcolm Turnbull, Minister for Defence Marise Payne and Minister for Defence Industry Christopher Pyne were at the event to welcome the aircraft.
Prime Minister Turnbull said the arrival of the JSFs was a great day for engineering, for innovation and for Australian enterprise.
The visiting aircraft were the first two Australian JSFs to be delivered out of the 72 Australia has ordered.
The two aircraft were piloted by Wing Commander Andrew Jackson and Squadron Leader Andrew Bell.