| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Okeechobee |
A | Bernal | Abner-Cayton | Whipple | Sallette | 5:26.40 | Sebring Relay Meet 2025 |
| 3 | Clewiston |
A | Johnson | Scuderi | Miscaide | Stephens | 4:52.19 | 65th Annual Edison Relays |
| 4 | Jensen Beach |
A | Thurber | Feurer | Bhatt | Brown | 4:43.04 | Bill Wilson Invitational 2025 |
| 5 | Jensen Beach |
B | Schmidt | Fischer | Glover | Laurent | 4:43.04 | Bill Wilson Invitational 2025 |
| 6 | Calvary Christian (PtStL) | A | Morales | Clark | Gonzalez | Brown | 5:01.42 | Bulldog Invitational 2025 |
| 7 | Treasure Coast Classical | A | Smith | Griffin | Russotti | Wolf | 5:28.82 | MCHS Fesh/Soph 2025 |