The towering lock sustained the injury early in the second half of the Queensland Reds' 13-12 win over the ACT Brumbies in a Super Rugby match in Canberra on Saturday.
Despite the injury, the skipper joined up with the Australia squad and coaching staff in Sydney on Sunday and will undergo a scan on Monday but is expected to miss out on the Tests.
Horwill's absence will force the Wallabies to name a new captain when they face Scotland on June 5 in Newcastle before the first of three Tests against Six Nations champions Wales on successive weekends from June 9.
Scrum-half Will Genia and openside flanker David Pocock are the likely candidates to lead the Tri-Nations champions in the absence of Horwill.
Fly-half Quade Cooper was included in the 39 despite playing only 120 minutes of rugby since a serious knee injury sustained at last year's World Cup.
Cooper had some poor moments in the Reds win over the Brumbies on Saturday and his inclusion in the squad, which features 15 uncapped players, was a result of an injury doubt over Melbourne Rebels playmaker Kurtley Beale, the Wallabies said.
Beale, who injured his shoulder playing for the Rebels in Wellington on Saturday night, was selected but it was unclear as to when the full-back-turned-fly-half will be fit to play.
Cooper's inclusion was also helped by Beale's team mate, utility back James O'Connor, also suffering an injury. The goalkicker is out of the squad because of a lacerated liver.
With a final round of Super Rugby games to be played before the international break, coach Robbie Deans will be hoping no more of his squad suffer injury before he names his team on Sunday for the Scotland Test.
"We can't finalise a side for that match until all of the games are played, and we are in a position to assess all of the players," Deans said in a statement.
"Obviously we have preliminary ideas but the selection template is a living document.
"It can, and probably will change, as a result of events through the final round of Super Rugby before the international break."
There was also no place in the 39 for former captain Rocky Elsom, with Australian media speculating that the 29-year-old flankers' international career was over after he was overlooked by Deans.
