| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Glades Day |
A | Alvarez | Baez | Sturgis | Bailey | DNS | Glade Central Invitational 2024 |
| 3 | Glades Central |
A | Pinto | Forbes | Blaise | Stewart | 52.04 | Pahokee Anquan Boldin Invitational 2025 |
| 4 | Pahokee |
A | Jones | Johnson | White | Washington | 49.42 | FHSAA 1A Region 4 |
| 5 | Inlet Grove |
A | Harper | Steward | Gibson | Sharpe | 51.68 | John I Leonard Invitational 2 |
| 6 | Somerset College Prep | A | Bourne-Morgan | Chase | Wright | Miller | 52.45 | Falcon Classic 2024 |