| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | John Carroll |
A | 45.29 | Benjamin Invite #1 2025 (Sanctioned) |
| 2 | Sebastian River |
A | 44.17 | Lincoln Knowles Invitational 2024 (FHSAA Sanctioned) |
| 3 | Fort Pierce Westwood |
A | 43.57 | FHSAA 3A District 13 |
| 4 | Vero Beach |
A | 42.27 | FHSAA 4A District 10 |
| 5 | Centennial |
A | 42.36 | Royal Palm Beach Meet 2025 |
| 6 | Fort Pierce Central |
A | DNF | Cobra Relays 2024 |
| 7 | Jensen Beach |
A | 45.15 | Benjamin Invite #1 2025 (Sanctioned) |
| 8 | Odyssey Charter |
A | 46.47 | Heritage Invitational |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 4 | Lincoln Park |
A | DNS |