| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Martin County |
A | Dennis | Gilbride | Kideys | St. Juste | 44.83 | South Fork Meet #1 2024 |
| 2 | Fort Pierce Central |
A | Louinssant | Watts | Victorin | Filsaine | 43.63 | Cobra Relays 2024 |
| 3 | Jupiter |
A | Madden | Grashof | Sirmans | Fassberg | 42.65 | FHSAA 4A Region 3 |
| 4 | Centennial |
A | Johns | Dorelus Jr. | Polynice | Jean-Pierre | 40.96 | Bill Wilson Invitational 2025 |
| 5 | Blanche Ely |
A | Warren | Parchment | Castriota | Hollis | 42.04 | FHSAA 3A District 14 |
| 6 | Benjamin School |
A | Etienne | Reddick | Di Leo | Scaperotto | 42.86 | Panther Relays 2025 |
| 7 | Wellington |
A | Spence | Benedetti Alizo | Borgella | Thomas | 44.32 | FHSAA 4A District 11 |
| 8 | Jupiter Christian |
A | McMillan | Fletcher | Baldwin | Mirabella | 46.45 | St Andrews Varsity |