| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Mulberry |
A | DNS | |
| 2 | DeSoto County |
A | 44.91 | Dunbar Tiger Classic Invite |
| 3 | Sebring |
A | 43.49 | Avon Park Meet #1 2025 |
| 4 | Avon Park |
A | DNF | Sebring Relay Meet 2025 |
| 5 | Hardee |
A | 45.21 | Larry Richardson Memorial Invitational |