O'Keefe, who impressed selectors with a 10-wicket haul in Australia's only practice game in Sri Lanka, had to leave the field in the middle of his 17th over of the second innings on day three yesterday.
"Initial assessment indicates a moderate grade hamstring injury which means it is highly unlikely that he will be available for the remainder of the Test series," Australian physiotherapist David Beakley said in the Cricket Australia statement.
Left-arm spinner Jon Holland has been called up to the Australian squad to replace O'Keefe, who picked up three wickets in the match, which looks evenly poised, before getting injured.
"It is incredibly disappointing for Steve to get this injury and I really feel for him. He was bowling well in the Test and had caused some trouble to Sri Lanka's top order," said Australian selector Rod Marsh.
"The selectors have decided to go for a like-for-like replacement for Steve, in Jon Holland.
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