| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Terry Parker |
A | 44.67 | 44.67 2024 Gateway Conference Track Championships |
| 3 | Westside |
A | DQ | 43.75 2024 Donnell Butler Invitational |
| 4 | Riverside |
A | 43.34 | 43.34 Adrain Mann Wolfpack Classic %2841st Annual%29 |
| 5 | Stanton |
A | 43.74 | 43.74 2024 Gateway Conference Track Championships |
| 6 | Orange Park |
A | 44.05 | 44.05 Don Hall meet |