The Repsol Honda rider, the championship leader and winner of four of the seven grands prix this season, recorded a time of one minute, 48.034 seconds.
Stoner finished almost half a second faster than closest challenger American Ben Spies, winner of last weekend's Dutch TT at Assen.
Italian Marco Simoncelli, who earned his second pole of the season in Assen and was quickest in Friday's first free practice session, will start third behind Stoner and Spies on the grid in his home race.
Another Italian, Andrea Dovizioso, is at the front of the second row, alongside Jorge Lorenzo, the reigning world champion who is second in the overall standings, and American Colin Edwards.
Stoner, who now has five poles this season, said on the BBC website: "We made one change on the bike from Saturday morning and it worked out really well.
"To set this time with tyres that had already put in 10 laps is very encouraging for the race."
Spies added: "We've got a good bike, but not yet good enough. Everyone expects a lot of me, I'm aiming for a podium finish."
Dani Pedrosa, last year's winner at Mugello after leading from start to finish, was eighth in qualifying on his return from a three-race lay-off after breaking his collarbone in a clash with Simoncelli at the French Grand Prix in May.