| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | St. Edward's |
A | Taft | Cocks | Dambournet | Perrin | 49.46 | Benjamin Invite #1 2025 (Sanctioned) |
| 4 | Jupiter |
A | Sirmans | Fassberg | Grashof | Madden | 42.65 | FHSAA 4A Region 3 |
| 5 | Jupiter Christian |
A | Fletcher | Mirabella | Fauntleroy | Zuniga | 46.45 | St Andrews Varsity |
| 6 | Treasure Coast Classical | A | Goldberg | Freed | Tomasini | Deyarmond | DNS | Jensen Beach / Chandler Wealth Invitational 2024 |