| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Lake Zurich North | A | Murray, Sam | Sullivan, Liam | Rampino, Anthony | Schagrin, James | 1:01.00 | |
| 2 | Grayslake | A | Poprawski, John | Dominguez, Raul | Chamberlain, Zach | Adams, Jeremy | 54.00 | |
| 3 | Barrington-Prairie Campus | A | Alvarez, Roberto | Barron, Nick | Kennedy, Nolan | Garcia, Andy | 51.78 | |
| 4 | Hawthorn South | A | Han, Krystofer | Franciosi, Jacopo | Kim, Sean | Shin, Dan | DQ | |
| 5 | Lake Zurich South | A | Graunke, Danny | McCall, Aidan | Remez, Tyler | Beaupre, Luke | 51.20 | |
| 6 | Hawthorn North | A | Hackett, Colin | Shores, Matthew | Thames, Matthew | Burrows, Nick | DNS | |
| 7 | Barrington-Station Campus | B | Ridgway, Ben | Keenan, Connor | Krueger, Alex | Darlington, Eric | 54.60 |