| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Trinity Prep |
A | Burgos | Hennebery | Del Vecchio | Macau | 4:40.73 | 4:40.73 Citrus League Championship 2024 |
| 4 | Geneva School |
A | Newhartz | White | Blanton | Shepherd | 4:28.91 | FHSAA 1A District 7 |
| 5 | Circle Christian |
A | Moury | Moury | O'Brien | Kirchberg | 4:36.68 | 4:36.68 %232 Mount Dora Developmental Meet |
| 6 | Trinity Christian (Deltona) |
A | McMillan | Bordner | Sanchez | Easter | NT | |
| 7 | Geneva School |
B | NT |