| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | John Carroll |
A | 4:26.78 | FHSAA 1A Region 4 |
| 3 | Fort Pierce Central |
A | 4:18.48 | FHSAA 4A District 10 |
| 4 | Vero Beach |
A | 4:02.08 | Tiger Invitational |
| 5 | Sebastian River |
A | 4:12.25 | FHSAA 3A Region 4 |
| 6 | Centennial |
A | 4:20.48 | 28th Annual Warrior Invitational 2024 |
| 7 | Jensen Beach |
A | 4:48.36 | Falcon Classic 2024 |