| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | DeSoto County |
A | 57.49 | HARDEE HS INVITATIONAL |
| 3 | Hardee |
A | 54.27 | Sebring Relays 2024 |
| 4 | Avon Park |
A | 54.61 | Lake Placid Meet #2 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Clewiston |
A | 53.11 | Antone Smith Muck City Invitational 2024 |
| 3 | Moore Haven |
A | 52.30 | South Fork Meet #1 2024 |
| 4 | Sebring |
A | 53.10 | Lake Placid Meet #3 |
| 5 | Lake Placid |
A | 53.75 | Lake Placid Meet #2 |