| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Treasure Coast Classical | A | Sullivan | Prestia | Apfel | Hook | 1:00.96 | MCHS Sarge Invitational 2025 |
| 2 | Tradition Prep | A | Delevery | Falcon | Derius | Parrel | 54.22 | Atlantic HS Last Chance |
| 3 | South Fork |
A | Marrone | Pepsidero | Padgett | Holmes | 52.70 | FHSAA 3A Region 4 |
| 4 | Dwyer |
A | Jean | Smith | Verdecia Perez | McClean | 48.83 | FHSAA 4A Region 2 |
| 5 | Martin County |
A | Osdon | Edwards | Prusenko | Perez | 50.18 | FHSAA 4A Region 2 |
| 6 | Moore Haven |
A | Myers | Hunter | Myers | Faison | 53.35 | FHSAA 1A Region 3 |
| 7 | Jensen Beach |
A | Trimble | Laurent | Peruski | Melda | 54.89 | Benjamin Invite #1 2025 (Sanctioned) |
| 8 | St_Andrews_Episcopal |
A | Roccasecca | Brown | Essoh | Hinote | NT |