Seeley clocked 115.836mph to head the session by 0.463s from Tobermore man Adam McLean on the McAdoo Kawasaki (115.644mph).
Seeley is a 12-time winner in the class and will be setting his sights on a milestone 25th success on the IFS Yamaha R6 after leaving the event empty-handed in 2019, when he failed to register a win for the first time since his maiden victory in 2008.
Third went to the consistent Davey Todd on the Milenco by Padgett’s Honda, who was only 0.644s behind Seeley, while Michael Dunlop was also in the frame on his MD Racing Yamaha, one second further back.
Dean Harrison (DAO Racing Kawasaki) and James Hillier (OMG Yamaha) finalised the first six, while Lee Johnston slots into seventh on the combined times on his Ashcourt Racing Yamaha after recording his fastest lap on Tuesday.
Magherafelt rider Paul Jordan produced a strong performance on the PreZ Racing Yamaha to go eighth quickest overall ahead of fellow Irish road racer Michael Sweeney on his EM Building Yamaha.
The first Supersport race takes place on Thursday evening along with the opening Superstock and Supertwin events, with roads closed from 5pm to 9pm.
1 A Seeley (Yamaha) 115.836mph
2 A McLean (Kawasaki) 115.644mph +0.436s
3 D Todd (Honda) 115.569mph +0.644s
4 M Dunlop (Yamaha) 115.119mph +1.736s
5 D Harrison (Kawasaki) 115.074mph +1.845s
6 J Hillier (Yamaha) 114.43mph +3.452s