| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | DeSoto County |
A | NT | |
| 2 | Avon Park |
A | DNS | Avon Park Meet #1 2025 |
| 3 | Sebring |
A | 52.64 | Blue Devil Invitational (Regio |
| 4 | Hardee |
A | 53.58 | Larry Richardson Memorial Invitational |
| 5 | Lake Placid |
A | DNF | Sebring Relay Meet 2025 |