Moments after Alessandro Pier Guidi set an impressive 3m25.412s in the #51 Ferrari 499P with around 15 minutes to go, despite getting slightly held up later in the lap by a GTE Am Aston, he was usurped by the sister #50 car of Antonio Fuoco - who posted a 3m25.213s to grab the top spot.
The early pace was set by the #7 Toyota of Kamui Kobayashi, who put in a 3m25.485s right at the very start of the session - which ultimately left him 0.3s behind the Ferraris, and fractionally ahead of Brendon Hartley in the sister #8 GR010 HYBRID.
Next up in the two-by-two formation from the leading manufacturers were two of the Porsche 963 LMDhs, Frederic Makowiecki in the #5 heading Felipe Nasr in the #75 as both were 0.6s behind the Ferraris.
The final two cars from the Hypercar class to make it through to the eight-car Hyperpole shootout on Thursday were the two Cadillac Racing V-Series.R LMDhs - Sebastien Bourdais in the #3, just ahead of Earl Bamber in the #2 and both less than a second off the pace.
Kevin Estre's #6 Porsche was the first car to miss out and will therefore start ninth - having missed the cut-off by almost a second.
He was in turn nearly half a second ahead of Mikkel Jensen in the best of the Peugeot 9X8 LMHs, who was a further 0.5s faster than team-mate Loic Duval.
The 16-car Hypercar field was then rounded out by the Action Express Caddy, the two Glickenhaus machines and the Vanwall Vandervell 680.
The Jota-entered Porsche 963 did not set a time in the session and will therefore start last following an electronic issue.
The hour-long qualifying segment began with two early red flags, the first caused by a collision between the #22 United Autosports LMP2 machine of Filipe Albuquerque and the #36 Alpine-run ORECA of Matthieu Vaxiviere when they were jostling for position into the final chicane.
The session had only just been restarted when it was halted again, this time by the #37 Cool Racing LMP2 entry of Malthe Jakobsen skating into the Indianapolis barriers.
For the majority of the session, the LMP2 leaderboard was headed by the #28 Jota entry of Pietro Fittipaldi, his 3m34.751s good enough to beat the #41 WRT-entered ORECA of Louis Deletraz by just two-thousandths of a second.
There was a far larger gap of 0.04s back to the next of the cars, the #63 Prema ORECA of Mirko Bortolotti, who had been the initial pacesetter.
However, there was late drama in the GTE Am leaderboard.
Charlie Eastwood's TF Sport Aston Martin Vantage had topped the times for much of the hour before a flurry of late improvements.
First up, with just four minutes to go, Nicky Catsburg in the sole Corvette C8.R emerged from the pits following repairs after Nico Varrone's Tertre Rouge practice crash to shoot to the top of the times on his very first flying lap.
But he was shuffled back to an eventual third following a pair of late efforts from Ferrari drivers.
Davide Rigon in the #54 AF Corse 488 looked to have snared top spot before he was pipped by the #83 Richard-Mille-backed sister car of Alessio Rovera moments after the chequered flag went out.
Cars progressing to Hyperpole:
Hypercar
