| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Cocoa Beach |
A | Manning | Daly | Cerveny | Reyes | DNS | FHSAA State Track & Field Championships (2A/Para-Athlete) |
| 2 | Pineapple Cove |
A | Guerra | Lucas | Guardie Valverde | Saunders | 8:24.90 | FHSAA State Track & Field Championships (1A) |
| 3 | Holy Trinity |
A | Nazareno | Martinez | Penny | Darlington | 8:25.00 | |
| 4 | Jensen Beach |
A | Cessna | Galdys | Belville | Acosta | 8:42.57 | The Lincoln Knowles Invitational 2025 |
| 5 | Viera |
A | Pasanen | Taylor | Valderrama | Islas | 8:45.00 | |
| 6 | Heritage |
A | Rhymes | Berrios | Peller | Cruz de Leon | 8:45.86 | FHSAA 4A District 7 |
| 7 | Sebastian River |
A | Hewitt | Perez | Sanchez | Gamez | 9:29.78 | St. Cloud Invitational (Qualifier) |
| 8 | West Shore |
A | Estevez | Falls | Duong | Daut | 9:30.00 | |
| 9 | Melbourne |
A | Hanus | Fernandez | Pereira | Aguilar | 9:34.67 | Viera High School Invitational |
| 10 | Melbourne Central Catholic |
A | Carney | HUI | Green | Lastsinger | 9:45.00 | |
| 11 | Merritt Island Christian |
A | Allwood | Bush | Baggett | Schermbeck | DNS |