The championship leader spent much of the session batting for the top spot with his sometime Moto2 title rival Stefan Bradl.
The German, who sealed a new LCR Honda deal at the start of the weekend, managed to depose factory Honda man Marquez from first place several times in the first part of the morning.
In the end Marquez stamped his authority on the session with a 1m40.781s, putting him half a second clear of the pack at that pack.
Cal Crutchlow ultimately emerged as his closest rival. A 1m41.081s from the Briton as he took the chequered flag moved his Tech 3 Yamaha to second, exactly three tenths off Marquez.
World champion Jorge Lorenzo had a spell on top and ultimately beat Bradl to third place on his Yamaha.
Lorenzo's team-mate Valentino Rossi edged out Alvaro Bautista and Dani Pedrosa for the final top-five place.
Nicky Hayden was top home rider in ninth, just behind his Ducati team-mate Andrea Dovizioso.
Hayden's countryman Ben Spies began his MotoGP comeback with a solid 11th place.
The 12 prototypes filled the top 12 positions, meaning CRT pacesetter Aleix Espargaro had to settle for an unusually low 13th.
Yonny Hernandez was the second-quickest CRT for Paul Bird despite having to abandon his primary bike following a minor fall.
The biggest accident of the session befell Karel Abraham. The Czech rider is being treated for a potential shoulder injury having landed badly in a high-side at Turn 10 early on.