A police spokeswoman said emergency services were called to the crash at Kawarau Bridge at about 11.45am.
A total of five people suffered injuries in the three-vehicle crash.
A Hato Hone St john spokesman said two people were seriously injured, one suffered moderate injuries and two more minor injuries.
The two seriously injured patients were flown to Dunedin Hospital while the others were either treated at the scene or taken by ambulance to Lakes District Hospital.
A Fire and Emergency NZ spokesperson said three people were trapped when firefighting crews first arrived.
Crews from Frankton, Arrowtown and Queenstown responded.
The road was fully blocked in both directions for about 2.5 hours while emergency services worked at the scene.
It reopened under traffic management with a 50kmh speed limit at about 2.15pm.
Motorists are advised to expect delays and take extra care along the route.
The Serious Crash Unit has been advised.
