| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Hardee |
A | Graham | Pierre | Blanden | Jackson | 53.47 | Mike Williams Classic @ Lake Region 2025 |
| 4 | DeSoto County |
A | Tyler | Reed | Henry | Campbell | 52.06 | Sebring Meet #2 2025 |
| 5 | Avon Park |
A | Davi | Berlin | Glabach | Simmons | 53.03 | Sebring Meet #2 2025 |
| 6 | Lake Placid |
A | Dillon | Legree | Claudio | Carlini-Smith | 59.58 | Sebring Meet #2 2025 |