| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Lake Highland Prep |
A | Bacon | DeMartino | Williams | Kleiman | 54.71 | Geneva Classic 2025 |
| 3 | Geneva School |
A | Lindsay | Newhartz | Flores | Duvra | 52.58 | Hagerty Track and Field Invitational 2025 |
| 4 | Menendez |
A | Oxendine | Watkins | Smith | Moore | 51.12 | |
| 5 | Bishop Moore |
A | Cantero | Sullivan | Suffran | Job | DNS | ROYAL INVITATIONAL @ The First Academy 2025 |
| 6 | South Walton |
A | De La Cruz | Castle | Fleeman | Rupe | 52.65 | Seahawks Open |
| 7 | Central Florida Christian |
A | Andre | Hastings | Noel | Piroupo | 1:00.97 | Geneva Classic 2025 |