| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Windermere |
B | Rossi | Griffin | Alexander | Perkett | DNS | |
| 3 | Colonial |
A | Jones | Castaneda | Batres | Jordan | 44.50 | |
| 4 | Windermere |
A | Riley | Jones | Paugh | Davis | 41.43 | |
| 5 | University (Orlando) |
A | Grullon | Phillips | Hinds | Dewberry | 43.91 | FHSAA 4A Region 1 |
| 6 | Innovation_Orlando | A | Caballero | Davila | Delarosa | Reyes | 45.40 | Ocoee Invitational |
| 7 | Innovation_Orlando | B | Hubbard | Harris | Espinal Ortega | Ramero | DNS |