The 30-year-old sustained the issue while playing for Denmark against Switzerland, playing just 16 minutes of the match before having to be taken off.
Harder was unsurprisingly absent from Sunday's 3-0 Women's Super League win over Tottenham Hotspur at Stamford Bridge, and Chelsea have now conceded that the injury will rule her out for a while.
A statement released on chelseafc.com confirmed that the forward had undergone successful surgery, but they did not place a timeframe on her recovery period.
"Following a hamstring injury sustained while representing her native Denmark during the international break, Pernille has been reviewed at Cobham by the Chelsea medical team," the statement read.
"Following that review and further discussion with a specialist, Pernille has undergone surgery and is expected to be sidelined for a significant period.
Pernille will now begin a rehabilitation programme with support from the club's medical team. Everyone at Chelsea sends their best wishes to Pernille in her recovery."
Harder uploaded an image of her smiling from her hospital bed following her surgery, and the Chelsea attacker believes that she will come back in "better shape" following the procedure.
"Saturday I underwent a successful operation on my hamstring. A time with recovery and rehabilitation now awaits before I can return to the pitch," Harder wrote.
"It's never fun when those things happen but I'm ready to work hard and take on the challenge. No matter how hard it is with the injury, I will use it as an opportunity to come back in even better shape when I'm out on the other side.
"I feel lucky to be surrounded by so many talented people who I know will help me on the road back to recovery so I feel very safe ❤️."
Harder had enjoyed a positive start to the 2022-23 campaign with Emma Hayes's side, scoring six goals and providing two assists in six games across the Women's Super League and Women's Champions League.
The former Wolfsburg attacker won her second successive WSL title with Chelsea last season and placed second in the 2018 Ballon d'Or Feminin behind Ada Hegerberg.