After being stepped on, O'Neill went down twice then came off the field just 36 minutes into New York City FC's 2-0 loss to DC United on Sunday (Monday AEST).
The news will send waves through Socceroos camp, given O'Neill, looking to play in his first World Cup finals, has been coach Tony Popovic's first-choice defensive midfielder along with Jackson Irvine.
More recently the likes of Paul Okon Jnr and Max Balard have also paired up with O'Neill, 27, who has started Australia's past seven games and has 29 caps.
The Socceroos will hope O'Neill's injury is on the less serious end of the scale, given the short lead-in to the World Cup, with Australia's first game on Sunday June 14.
O'Neill's NYCFC coach confirmed, much like Irvine, he had already been playing with an ankle problem.
"Yeah he has an ankle issue," Pascal Jensen said.
"But he plays with some issues in his ankle all the time, he doesn't know any better, in fact. So that is something that we're aware of.
"But he had a moment in the game, I think it was five or six minutes before we took him off, he had a moment where he tried to turn and Aiden is a player that plays on the half turn always, either left or right, doesn't matter.
"But his ankle just locked and had too much pain to continue, he tried for a couple minutes and I know for a fact that Aido is not a guy that just steps out unnecessarily."
O'Neill's teammate Kai Trewin played a full game, as did fellow Aussie defender Kye Rowles for DC United.
Popovic is due to speak to media on Monday afternoon in Sydney.
The Socceroos have a training camp in Sarasota, then play a friendly against Mexico in Los Angeles on May 31 - the last game before Popovic selects his squad on June 1 - before relocating to their Bay Area base.
Australia then play Switzerland on June 7 in San Diego, their final warm-up game.
The Socceroos face Turkey in Vancouver on June 14, the United States in Seattle six days later then Paraguay in Santa Clara on June 26 (all dates AEST).
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