The Cats ended a three-match losing run with a comprehensive 15.12 (102) to 11.9 (75) win over St Kilda on Thursday night, strengthening their grip on a finals spot.
But post-match celebrations were tempered by the sight of Holmes on crutches and with his right foot in a protective boot.
Geelong have confirmed an ankle fracture and syndesmosis injury, with Holmes to undergo surgery on Friday.
The speedster will likely miss the rest of the home-and-away season, but Geelong will provide a return-to-play timeframe following surgery.
Holmes was clearly in pain and had to be helped off the ground during the final quarter after his right leg twisted underneath him.
Geelong coach Chris Scott "covered my ears" when medical staff explained the outlook on Holmes.
Geelong (10-8) are already without key forward Jeremy Cameron, who had surgery to repair an AC joint and is expected to be sidelined for four to six weeks.
"Obviously it's frustrating, but it is the game," Scott said.
"If you asked me a couple of weeks ago who do you really not want to lose, (Jeremy) Cameron and Holmes would be up there."
Shaun Mannagh (hamstring tightness) also failed to finish the match as Geelong surged as much as 47 points clear before conceding the final three goals.
Mark Blicavs, Jake Kolodjashnij and Jack Martin will all be available against Melbourne next week after they were rested against the Saints.
Tanner Bruhn could also return after missing with a neck injury.
St Kilda (8-10), meanwhile, could drop out of the wildcard places by the end of the round, but are set to welcome back spearhead Max King from his two-year absence.
King has all but completed his conditioning program after a raft of injury setbacks and is almost certain to play his first AFL game in more than two years against North Melbourne next round.
"Saints fans, get ready for him playing. Yeah, he'll be playing," St Kilda coach Ross Lyon said.
"He's flying, to be honest. He's got to get through the week but he's in pretty good nick and he's keen to play, and that's exciting for us.
"If he's gets through what we think he's getting through on the weekend and then into Sunday, he'll play."
Lyon said ruck-forward Tom De Koning is also a chance to return from a rib injury to play against the Kangaroos.
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