| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | St. Edward's |
A | Jackson | Collentrelle | Leibik | Dambournet | NT | |
| 2 | Suncoast |
A | Caple | Burnett | Campbell | Normil | DNS | Clyde Harris Invitational at Boynton |
| 3 | Spanish River |
A | Sainmervil | Smalls | Frederick | Goulbourne | 45.25 | |
| 4 | Jupiter |
A | Jackson | Fassberg | Madden | Grashof | 42.65 | FHSAA 4A Region 3 |
| 5 | Benjamin School |
A | Etienne | Reddick | Winfield | Faniel | 43.17 | FHSAA State Track & Field Championships (1A) |
| 6 | Jensen Beach |
A | Warburton | Fell | Chrysler | Porter | 45.32 | South Fork Meet #2 2024 |
| 7 | John Carroll |
A | Monaco | Bradley | Summerall | Coleman | 45.90 | SLC Championships 2024 |