| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Lakewood Ranch |
A | Bernardes | Antoine | Criniti | Johnson | 56.00 | |
| 3 | Lemon Bay |
A | Mikulskis | Jackson | Strahan | Woitalewicz | 52.58 | 52.58 Sarasota County Track and Field Championship |
| 4 | Booker |
A | Byrd | Saunders | Saunders | St. Louis | 48.09 | FHSAA 2A Region 3 |
| 5 | Southeast |
A | Peterson | Robinson | Ramirez | Mobley | 51.10 | FHSAA 3A Region 3 |
| 6 | Bradenton Christian |
A | Stanley | Bell | Pierre | Gibson | 53.17 | 53.17 Sunshine State Athletic Association Championship 2025 |
| 7 | Braden River |
A | Valdez-Morales | Duprey | Anthou | Scott | 58.00 |