The benchmark set by LCR Honda rider Cal Crutchlow on Friday, a 1: 47.365s, was beaten early in the 45-minute session by Aleix Espargaro’s Aprilia, who topped the times with 1: 47.173.
That remained as the best time into the final minutes, when the order was completely reshuffled by a flood of fast times with soft tires.
Ducati rider Jorge Lorenzo was the first to beat Espargaro with 1: 46.911 minutes four minutes before the end.
But Marquez’s time was beaten with just 30 seconds left on the clock by Rossi, who suffered arm pain on his way to 11th and 12th in Friday’s sessions after his motocross crash last week.
The Yamaha rider set a time of 1: 46.543 minutes to end the session 0.322 seconds ahead of Marquez and 0.368 seconds over Lorenzo, who stayed third.
Alvaro Bautista placed his 2016 Aspar Ducati in fourth place, beating the second Yamaha from Maverick Vinales, who finished the session 0.429 seconds behind teammate Rossi.
Tito Rabat took full advantage of Marquez’s slipstream and finished sixth on board the Marc VDS Honda, followed by the remaining factory Ducatis from Michele Pirro and Andrea Dovizioso and the second Repsol motorcycle from Dani Pedrosa.
Espargaro’s early time was just enough for 10th place and the last automatic starting position in Q2.
Crutchlow, who crashed on his outlap at Turn 14, couldn’t improve his time on Friday and finished 12th ahead of the two Pramac Ducatis of Scott Redding and Danilo Petrucci, the Italian who crashed twice.
Andrea Iannone was the best of the Suzukis in 17th place, one place ahead of leading KTM rider Pol Espargaro.