All eyes were on the Yamaha of Lorenzo after he set the fastest-ever lap time at the track in FP3 with a 1:46.617, but Iannone bettered that mark in Q2 with his 1:46.489 earning Ducati their first pole position at Mugello since 2007.
The other Ducati of Andrea Dovizioso will also start on the front row with Lorenzo, who is looking for a hat-trick of wins in May following his victories at Jerez and Le Mans, sandwiched in between.
The second row will be occupied by Cal Crutchlow from CWM LCR Honda, Aleix Espargaro on the Suzuki and wildcard rider Michele Pirro.
Dani Pedrosa on the Repsol Honda continued his recovery from surgery and was seventh fastest while the second Yamaha of Valentino Rossi will join him on the third row.
If you were looking for the name Marquez, well, he will start in P13 following a disastrous day.
There was the unusual sight of the Repsol Honda rider in Q1 after he could only finish P11 in FP3 and he got it wrong at the first corner, the same place where he crashed in FP4, on his first flying lap.
The second time of asking the defending World Champion produced a much better effort of 1:47.420, which was 0.018s off Aleix Espargaro's fastest Q1 time, and the team elected not to send him out again.
It proved to be the wrong call as Yonny Hernandez on the Pramac Racing improved on Espargaro's time with his final effort to bump Marquez out. The Spaniard could be seen hitting a table in the garage and then took his cap off and threw it on the floor.
Meanwhile, there was contrasting scenes in the Pramac Racing garage after Hernandez's brilliant effort and the Colombian will start P12 following a solid Q2 session.
1 Andrea IANNONE Ducati Team 1'46.489
2 Jorge LORENZO Movistar Yamaha 0.095
3 Andrea DOVIZIOSO Ducati Team 0.121
4 Cal CRUTCHLOW CWM LCR Honda 0.168
5 Aleix ESPARGARO Team SUZUKI 0.365
6 Michele PIRRO Ducati Team 0.381
7 Dani PEDROSA Repsol Honda Team 0.386
8 Valentino ROSSI Movistar Yamaha 0.434
9 Maverick VIÑALES Team SUZUKI 0.445
10 Pol ESPARGARO Monster Yamaha Tech 3 0.561
11 Bradley SMITH Monster Yamaha Tech 3 0.601
12 Yonny HERNANDE Octo Pramac Racing 0.934
13 Marc MARQUEZ Repsol Honda Team 0.123
14 Danilo PETRUCCI Octo Pramac Racing 0.380
15 Hector BARBERA Avintia Racing 0.861
16 Stefan BRADL Athinà Forward Racing 0.930
17 Scott REDDING EG 0,0 Marc VDS 1.003
18 Loris BAZ Athinà Forward Racing 1.016
19 Nicky HAYDEN Aspar MotoGP Team 1.181
20 Karel ABRAHAM AB Motoracing 1.249
21 Alvaro BAUTISTA Aprilia Racing Team Gresini 1.360
22 Mike DI MEGLIO Avintia Racing 1.386
23 Jack MILLER CWM LCR Honda 1.455
24 Eugene LAVERTY Aspar MotoGP Team 1.521
25 Alex DE ANGELIS E-Motion IodaRacing Team 2.081
66 Marco MELANDRI Aprilia Racing Team Gresini 4.274