| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Treasure Coast Classical | A | Ake | Freed | Kalinski | Tomasini | 1:02.60 | MCHS Fesh/Soph 2025 |
| 2 | St. Edward's |
A | Colontrelle | Cocks | Dambournet | Donohoe | DNS | Benjamin Invite #1 2025 (Sanctioned) |
| 3 | Clewiston |
A | Mason | Moody | Pope | Harshaw | 44.30 | Kings Academy Varsity 2 |
| 4 | Jensen Beach |
B | Avril | Danvers | Campion | Pino | 42.05 | Bill Wilson Invitational 2025 |
| 5 | Jensen Beach |
A | Warburton | Fell | Porter | Chrysler | DQ | Bill Wilson Invitational 2025 |
| 6 | Okeechobee |
A | Evans | Hamilton | Stewart | Brifil | 45.29 | Sebring Relay Meet 2025 |
| 7 | Calvary Christian (PtStL) | A | Alexis | Posten | Thompson | Gonzalez | 57.22 | Bulldog Invitational 2025 |
| 8 | Braddock |
A | Duran | Garcia | Jacobo | Manso | DNS |