| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Tradition Prep | A | Parrel | Josil | Derius | Delevery | 53.15 | Jensen Beach Invitational 2026 |
| 3 | Fort Pierce Westwood |
A | Blaise | Brown | Jackson | Villegas | 49.45 | Bill Wilson Invitational 2025 |
| 4 | Benjamin School |
A | Morse | Arendts | Reddick | Marshall | 48.11 | FHSAA State Track & Field Championships (1A) |
| 5 | Centennial |
A | Miller | Sinclair | Chery | Green | 49.39 | Louie Bing Invitational |
| 6 | Treasure Coast | A | Smith | Smith | Gammon | Sama | 50.11 | The Lincoln Knowles Invitational 2025 |
| 7 | St. Edward's |
A | Cannon | Neumann | Perez | Jeune | 55.04 | FPC Fresh/Soph Meet 2025 |