| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 4 | Poinciana |
A | Lee | Narcisse | Mendez | Morris | 3:28.50 | FHSAA 4A Region 2 |
| 5 | Winter Park |
A | Armstrong | Gardner | Knight | Sutton | 3:21.13 | FHSAA State Track & Field Championships (4A / Para-Athlete) |
| 6 | Edgewater |
A | Senatus | Edwards | Casanas | Cleveland | 3:28.10 | FHSAA 3A District 6 |
| 7 | Deltona |
A | Castro | Hutton | Degraff | Martinez | 3:49.58 | 2024 George Janan Spruce Creek Invitational |