The 27-year-old rider, who is going for his sixth consecutive world title in the premier class, clocked 1:31.002 on his Yamaha around the tight and turning four kilometre circuit.
The Italian will be joined by Ducati duo Troy Bayliss and Loris Capirossi on the front row of the grid, while championship contender Nicky Hayden, the only man who can deprive Rossi of the title, will start on the second row of the grid on his Honda.
"Tomorrow is the most important race of the year," Rossi told Spanish television after claiming his fifth pole of the season.
"I will be fully concentrated from the first to the last minute. The bike is going very well and I feel comfortable."
American rider Hayden had led the championship for most of the season, however Rossi is now top after finishing second in Portugal earlier this month in a race that his U.S. rival failed to finish.
Rossi leads the title race by eight points, but Hayden can still snatch the championship if he wins in Valencia and Rossi finishes worse than second place.