| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Wellington |
A | Brooks | Carbocci | Weir | Ramirez | 51.52 | Wellington HS Invitational |
| 3 | Centennial |
A | Schmook | Hoskins | Mitton | Schmook | 51.13 | Bill Wilson Invitational 2025 |
| 4 | Benjamin School |
A | Dalton | Arendts | Reddick | Marshall | 49.83 | Benjamin Invite #1 2025 (Sanctioned) |
| 5 | Treasure Coast | A | Brown | Sama | Smith | Labbe | 50.06 | FHSAA 4A District 10 |
| 6 | Fort Pierce Central |
A | Simeus | Bolden | Stinson | Wilder | 51.20 | Cobra Relays 2024 |
| 7 | Jupiter Christian |
A | Fielder | Winslow | Winslow | Gephart | 57.96 | Pine Crest Panther Relays |