| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | DeSoto County |
A | 3:46.79 | Avon Park Meet #1 2025 |
| 3 | Sebring |
A | 3:36.27 | Sebring Relay Meet 2025 |
| 4 | Hardee |
A | 3:42.26 | Larry Richardson Memorial Invitational |
| 5 | Avon Park |
A | 3:56.65 | Frostproof - The Ridge Open 2025 |