| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Moore Haven |
A | DNS | |
| 2 | DeSoto County |
A | 5:08.85 | FHSAA 2A District 11 |
| 3 | Sebring |
A | 4:21.61 | FHSAA 3A Region 2 |
| 4 | Lake Placid |
A | 4:30.51 | FHSAA 2A District 11 |
| 5 | Avon Park |
A | 5:36.90 | Avon Park Meet #1 2025 |
| 6 | Okeechobee |
A | NT |