| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 3 | Odyssey Charter |
B | NT | |
| 4 | Titusville |
A | 50.86 | 2025 Merritt Island Relays |
| 5 | Cocoa |
B | 45.60 | FHSAA State Track & Field Championships (2A/Para-Athlete) |
| 6 | Cocoa |
A | 45.60 | FHSAA State Track & Field Championships (2A/Para-Athlete) |
| 7 | Odyssey Charter |
A | 54.31 | FHSAA 1A District 8 |