| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Cypress Creek (Wesley Chapel) |
A | Axcius | Barringer | Boateng | Jones | 55.00 | |
| 3 | River Ridge |
A | Mclean | Rivera | Testa | Kurpejovic | NT | FHSAA 3A District 8 |
| 4 | Wesley Chapel |
A | Denning | Denning | Smith | Joseph | 48.45 | 48.45 Coyote Invitational Cypress Creek Wesly Chapel |
| 5 | Land O'Lakes |
A | Wright | Davis | Stephens | Oneal | NT | FHSAA 4A Region 3 |
| 6 | Gulf |
A | Quarg | Green | Brown | Ward | NT | FHSAA 3A Region 2 |
| 7 | Fivay |
A | Gay | Riley | Rodriquez | Paxton | 55.36 | FHSAA 3A District 8 |