Flintoff, sidelined for the first two Tests of the series against West Indies, will have investigative surgery over the weekend and miss the remaining two Tests which begin on June 7 and 15.
His participation in the July and August three-Test series against India is also in doubt, although the England and Wales Cricket Board (ECB) said on Wednesday that the latest problem was not as bad as on previous occasions when he has been out of action for several months.
"Andrew Flintoff has undergone rigorous fitness testing over the past two days, most recently 30 minutes of net bowling on Tuesday, but has reacted with further discomfort in his left ankle," ECB chief medical officer Nick Peirce said in a statement.
"Despite intense treatment and rehabilitation, Andrew's ankle has continued to cause him discomfort when bowling at full capacity. As a consequence he will undergo an exploratory arthroscopy over the weekend.
"This current ankle injury is separate to the previous posterior impingement injury and while a comprehensive timescale of rehabilitation will be clearer following the operation, the recovery period is not expected to be as long as Andrew's previous surgery."
Flintoff underwent surgery in 2005 and 2006, the latter forcing him to miss the second half of the English season.
The ankle problem continued to hamper him during the recent Ashes whitewash by Australia.
"Obviously I'm bitterly disappointed to face another operation on my ankle as I really thought I was making progress over the past few weeks," Flintoff said.
"I've been working really hard on my game and the rest and rehabilitation appeared to be helping the ankle but after giving it a strong workout this week, it now seems an operation is the only option."