Ben McDermott has been brought into the squad as cover for Short, who had an operation on his right index finger on Monday.
The day before, the off-spinning allrounder had played in the third and final one-dayer against India.
Short had damaged the finger in the second one-day game in Adelaide, when attempting a slips catch.
He was patched up and made 74 as Australia won, but doctors decided surgery was needed.
Josh Philippe is a candidate to take his place in Wednesday's series opener in Canberra, but Australian officials remain hopeful Short will play some part in the five-match series.
Short batted at No.3 earlier this month in the series against New Zealand, a sign of Australia's new aggressive T20 approach.
That will face its sternest test ahead of next year's World Cup in the form of tournament hosts and reigning champions India.
Canberra will be hostile territory for the hosts when the sides begin their five-game series, with die-hard India fans set to transform a sold-out Manuka Oval into a sea of blue shirts.
Since their ignominious second-straight T20 World Cup group-stage exit in 2024, Australia have pioneered a more aggressive, fast-scoring style that has resulted in them winning 12 of their last 13 matches.
Mitch Marsh has been pivotal to that success, with his all-out-attack during powerplays at the top of the order helping set the foundations for Australia's winning run.
But the captain downplayed his role in the transformation.
"I think that's happened quite organically to be honest, just with the personnel we've got in the team," he told reporters on Tuesday.
"We had two World Cups where we didn't quite go all the way and I guess we spoke about wanting to change ourselves as a team to what we think can win us a World Cup. And as a batting unit we have played a lot more aggressively.
"I think that's been the nature of T20 cricket for a lot of teams over the last few years.
"We're not gonna get it right every time. We will fail. But if we're clear on how we wanna go about it, that gives us the best chance for success."
But India is a sterner test than Australia has faced since their style shift.
The tourists have won 17 of their past 19 T20 and are strong favourites to retain the World Cup trophy at home in 2026.
"I know that we've got eight games to go for us and we're really building nicely with the way we want to play and go about it," Marsh said.
"We feel great at the moment but we know there's going to be a big series coming up."
Australia coach Andrew McDonald said the team had to change things up after a couple of unsuccessful campaigns.
"Is it the style that can win a World Cup? We believe that," he said on Monday.
"Will we have to pivot and nuance it slightly, depending on whether we're in Colombo or Delhi or Kolkata. There's no doubt about that."
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