| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Glades Day |
A | Villarreal | Crawford | Wooten | Clark | 44.85 | Pahokee Anquan Boldin Invitational 2025 |
| 3 | Pahokee |
A | Fitzgerald | Edward | Jones | Stinfort | 43.24 | Pahokee Andre Powell Invitational |
| 4 | Glades Central |
A | Stinfort | Stinfort | Ray | Goring | 42.69 | Pahokee Anquan Boldin Invitational 2025 |
| 5 | Santaluces |
A | Watkins | Zennot | Michel | Gerald | 43.09 | Panther Relays 2025 |
| 6 | Royal Palm Beach |
A | Treasure-Walker | Williams | Smith | Rolle | DNS | Royal Palm Beach Meet 2025 |
| 7 | Forest Hill |
A | Emerson | Henricy | Josapha | Rossow | DNF | Wellington HS Invitational |