| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Out-of-Door | A | Moschella | Meyers | Clarke | Crisci | NT | |
| 3 | Hardee | A | Simon | Valdez | Delhomme | Pierre | 45.11 | Berkley Track and Field Invitational |
| 4 | Riverview (Sarasota) | A | Campbell | Wheeler | Galva-Perez | Lester III | 44.29 | |
| 5 | St. Stephens Episcopal | A | Brewer | Bosek | Winterhalter | Mohr | 44.86 | FHSAA 1A District 11 |
| 6 | Winthrop College Prep | A | Hebert | Boyle | Ramos | Pringle | NT | |
| 7 | Cardinal Mooney | A | Hassler | Silva | White | Scurry | NT |