| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Dwyer |
A | Hutchins | Brown | Baptiste | Lee | 42.92 | Lincoln Knowles Invitational 2024 (FHSAA Sanctioned) |
| 4 | Jupiter |
A | Jackson | Turchetta | Madden | Grashof | 42.65 | FHSAA 4A Region 3 |
| 5 | Palm Beach Gardens |
A | Lubin | Grant | Alexander | Butler | 42.89 | |
| 6 | Inlet Grove |
A | Fischbacher | Grantlin | Francis | Francis | 43.08 | FHSAA 2A District 13 |