| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | University (OC) |
A | 55.35 | Sanford-Seminole Open 2025 |
| 3 | East Ridge |
A | 50.97 | FHSAA 4A District 4 |
| 4 | Seminole (Sanford) |
A | 48.38 | FHSAA State Track & Field Championships (4A) |
| 5 | Mount Dora |
A | 49.79 | FHSAA 3A Region 2 |
| 6 | Hagerty |
A | DQ |