| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Melbourne Central Catholic |
A | Davis | Stenlake | White | Conlon | DNS | 2025 Merritt Island Relays |
| 3 | Heritage |
A | Contreras Hidalgo | Ferguson | Barrett Brown | Jean-Louis | DNS | Cape Coast Conference Championship 2025 |
| 4 | Titusville |
A | Freeman | McKinstry | McKinstry | Ray | 4:16.16 | Cape Coast Conference Championship 2025 |
| 5 | Cocoa |
A | Hearns | Thomas | Henderson | Madyun | DNF | Tiger Invitational |
| 6 | Mount Dora Christian |
A | Filetti | Krauss | Lemoyne | Krauss | DNS | FHSAA State Track & Field Championships (1A) |
| 7 | Holy Trinity |
A | Maaser | McCarley | Young | Stewart | 4:20.69 | Viera High School Invitational 2 |
| 8 | Fort Pierce Westwood |
A | Holmes | Smith | Jackson | Moody | DNS | Cobra Relays 2025 |