| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | DeSoto County |
A | 4:53.79 | FHSAA 2A Region 3 |
| 2 | Avon Park |
A | 4:44.85 | Avon Park Chet Brojek Track Invitational |
| 3 | Sebring |
A | 4:09.09 | FHSAA 3A Region 2 |
| 4 | Okeechobee |
A | 5:26.40 | Sebring Relay Meet 2025 |
| 5 | Hardee |
A | 4:45.00 | |
| 6 | Lake Placid |
A | DNS | Sebring Meet #2 2025 |