| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Baker County |
A | 52.99 | 52.99 Bronco Bob Invitational |
| 2 | Ed White |
A | 52.74 | 52.74 2024 Gateway Conference Track Championships |
| 3 | Terry Parker |
A | 50.70 | 50.70 2024 Gateway Conference Track Championships |
| 4 | Westside |
A | 48.32 | 48.32 2024 Gateway Conference Track Championships |
| 5 | Riverside |
A | 50.19 | 50.19 2024 Chandra Cheeseborough Invitational - High School |
| 6 | Orange Park |
A | 50.76 | 50.76 Ridgeview High School Invitational |
| 7 | Stanton |
A | 52.89 | 52.89 Bob Hayes High School Invitational |