| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Avon Park |
A | Greene | Virkler | Glabach | Chamberlein | 5:09.57 | Frostproof - The Ridge Open 2025 |
| 4 | Hardee |
A | Villa | Manriquez | Holley | Ramos | 4:27.75 | Avon Park Meet #1 2025 |
| 5 | DeSoto County |
A | Elabed | Hannon | Henry | Johnson | 5:03.98 | Sebring Relay Meet 2025 |
| 6 | Lake Placid |
A | Hernandez | Dussaman | Santiago | Towner | 5:29.94 | Sebring Meet #2 2025 |