| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Imagine School |
A | Ruth | Sigmon | Arrowood | Traub | 4:52.52 | Wally Keller Invitational |
| 3 | Sarasota |
A | Grajalez | Daverio | Cicilioni | Cardell | 4:14.15 | FHSAA 4A Region 2 |
| 4 | Venice |
A | Myers | Sweiderk | Potter | Robinson | 4:05.21 | Pepsi Florida Relays |
| 5 | Riverview (Sarasota) |
A | Morris | Johnson | Miller | Tenaglia | 4:09.53 | Wally Keller Invitational |
| 6 | Cardinal Mooney |
A | Compa | Murray | Calovich | Selke | DNS | FHSAA 2A Region 3 |
| 7 | Sarasota Military Academy |
A | Contreras | Guzman | Foss | Schlabach | 5:05.82 | ODA Thunder Classic 2025 |