| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Lake Worth |
A | Robinson | Bell | Lajeune | Chambers | 45.97 | Park Vista #1 2025 |
| 2 | Dr. Joaquin Garcia | A | Charlot | Sineace | McKay | Vilma | 44.75 | Park Vista #1 2025 |
| 3 | Park Vista |
B | Hoang | Philosthene | Marsh | McGuire | 44.10 | Park Vista #1 2025 |
| 4 | Pahokee |
A | Fitzgerald | Edward | Mitchell | Stinfort | 43.24 | Pahokee Andre Powell Invitational |
| 5 | Spanish River |
A | Toscano | Frederick | Smalls | Campbell | 43.66 | 2025 Coral Springs Invitational |
| 6 | Park Vista |
A | Thelamond | Audette | Darrigo | Villalba | 44.10 | Park Vista #1 2025 |
| 7 | Olympic Heights |
A | Louis | Lewis | Toribio | Araujo | DNS | St Andrews Varsity |
| 8 | Boca Raton |
A | Roseme | Sejour | Alfonso | Buchanan | NT |