World No.43 Popyrin was due to play Jiri Lehecka in the round of 32 on Tuesday but withdrew on Monday.
The 24-year-old had been dealing with a leg injury in his Brisbane International loss to Roman Safiullin last Wednesday.
In that match, Popyrin let a huge third-set lead and five match points slip in a gut-wrenching 6-7 (11-9) 6-4 7-6 (7-3) defeat.
Adelaide International organisers on Monday confirmed Popyrin's injury.
It is unclear how Popyrin's injury will affect his hopes of playing in the Open, where he made the third round in 2019, 2020 and 2023.
Earlier on Monday, several Australians were in action.
Alex Bolt beat Matthew Dellavedova 6-1 6-4 in their qualifying match to secure passage to the round of 32.
Adam Walton also earned a main draw spot with a 6-3 6-2 win over Bernabe Zapata Miralles and will next play Lehecka.
James McCabe lost his qualifier to Frenchman Arthur Rinderknech 6-4 7-5 but earned a lucky loser spot against Alexander Bublik.
Englishman Jack Draper kicked off the round of 32 on Monday evening by beating fifth seed Sebastian Baez 6-1 6-3.
8°C