Hopkins takes pole, injured Rossi 18th
Rossi will start in 18th and last position. He struggled to control his bike after injuring his hand on Thursday in a crash and will decide today whether to race.
Riding a Suzuki, Hopkins robbed Japan's Shinya Nakano of his first pole in the dying seconds of qualifying, run in dry conditions. Hopkins clocked one minute 36.411 seconds and Nakano, on a Kawasaki, 1:36.424.
"It's been a long time coming," said Hopkins.
"It's been second, second, third -- but it's a huge weight off my shoulders."
American Colin Edwards (Yamaha) was third and world championship leader, American Nicky Hayden, fourth on his Honda.
Rossi broke a bone in his right hand on Thursday and it swelled badly overnight.
The Italian managed just four laps in free practice on Friday and limped round the track in qualifying, before calling it a day to rest his hand before today's race.
He was nearly four seconds slower than Hopkins.
"My wrist really hurts," Rossi told reporters. "It's hard riding with a swollen hand, there is not much strength in it. "I'm taking painkillers … but I had to stop because of the pain."
Rossi won last weekend's Catalan Grand Prix which featured a pileup on the first corner. Ducati's Loris Capirossi hurt his chest in that crash and was obviously still suffering during qualifying on Friday.
The onboard camera showed the Italian holding his fist to his chest and breathing deeply as he went round the Assen circuit. Capirossi, second in the standings, qualified 15th.
Spain's Jorge Lorenzo qualified fastest for the 250cc race, lapping his Aprilia in 1:40.007.
Lorenzo is second in the standings behind Italian Andrea Dovizioso (Honda) who is also on the front row in third, behind San Marino's Alex de Angelis on an Aprilia.
In the 125cc, Mika Kallio of Finland, second in the standings on a KTM, took pole. World championship leader Alvaro Bautista of Spain was second on an Aprilia and Lukas Pasek of the Czech Republic (Derbi) third.