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World champion Rossi, who trails Hayden by 21 points with three races remaining this season, won the first psychological battle at Motegi, the Italian clocking a fastest lap of one minute 46.981 seconds.
Japan's Shinya Nakano was second quickest behind Rossi's Yamaha with a time of 1:47.315 on his Kawasaki.
American Hayden was only fifth fastest on his Honda in a time of 1:47.610.
Italian Marco Melandri, who won last weekend's rain-hit Australian race to keep his slim title chances alive, was third in a time of 1:47.449.
Hayden currently has 225 points in the championship with a surging Rossi on 204 as he bids for his sixth straight top-class title. Melandri and Spanish Honda rider Dani Pedrosa are on 193.
The MotoGP season closes with races in Portugal and Spain.