| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Lake Placid |
A | 5:46.70 | Avon Park Meet #1 2025 |
| 2 | DeSoto County |
A | 5:03.98 | Sebring Relay Meet 2025 |
| 3 | Hardee |
A | 4:27.75 | Avon Park Meet #1 2025 |
| 4 | Sebring |
A | 4:28.95 | Avon Park Meet #1 2025 |
| 5 | Avon Park |
A | 5:09.57 | Frostproof - The Ridge Open 2025 |