Emily Bates tore her ACL in the match against Western Bulldogs.
The Hawks will also be without Laura Stone (concussion) against North Melbourne on Friday, while Daisy Flockhart faces a late fitness test.
The 30-year-old Bates, who played her 100th game the week before her injury, will have surgery.
Three-time All-Australian Emily Bates will miss the rest of the AFLW season with a knee injury in a large blow to Hawthorn's chances of snaring a maiden premiership.
Scans have confirmed Bates tore the anterior cruciate ligament in her right knee when the leg twisted awkwardly in a tackle during the Hawks' win over the Western Bulldogs on Saturday.
The 30-year-old club captain also damaged the meniscus and will undergo surgery.
"This is clearly disappointing news for Emily," Hawks AFLW general manager Keegan Brooksby said.
"The playing group and wider club will support her through the recovery journey.
"We have no doubt she'll approach her rehab with the diligence and professionalism she's always been known for.
"Although Emily won't return to the field in 2026, she will continue to play an important off-field role in our program, as our captain."
Hawthorn face a large test against AFLW juggernaut North Melbourne in Cairns on Friday night without Bates and Laura Stone (concussion).
Fellow midfielder Daisy Flockhart also has to prove her fitness after suffering a knock to her chest against the Bulldogs.
"We've been playing different players through [the midfield] and trying to build some versatility in our side … we're confident in the players we'll have in there," Hawks coach Daniel Webster said post-match on Saturday.
"It'll be a good challenge next week with who we're playing.
"We'll probably have some sort of different look in there with different players and that's OK. We'll keep getting to work."
Hawthorn (3-2) sit eighth in their quest for another finals berth, while North Melbourne (5-0) are in familiar territory on top of the ladder.