| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Fort Pierce Central |
A | Lane | Bolden | Stinson | Simeus | 51.80 | Cobra Relays 2025 |
| 4 | Centennial |
A | Elma | Mitton | Sinclair | Schmook | 50.50 | SLC Championships 2025 |
| 5 | Lincoln Park |
A | Huskin | Brown | Whilby | Brianvil | 50.95 | Bill Wilson Invitational 2025 |
| 6 | St. Edward's |
A | Gayle | Jensen | Jeune | Perez | 55.43 | Viera High School Invitational |