| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 3 | Father Lopez |
A | 45.88 | Bishop Moore - Carl Friedland 2026 |
| 4 | Matanzas |
A | 43.89 | FHSAA 3A Region 1 |
| 5 | Freedom (Orlando) |
A | 44.10 | Freedom Last Chance 2025 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | DeLand |
A | 42.73 | 18th Annual George Janan Spruce Creek Invitational |
| 3 | Lake Mary |
A | 42.08 | FHSAA 4A District 2 |
| 4 | Seminole (Sanford) |
A | 42.03 | 18th Annual George Janan Spruce Creek Invitational |
| 5 | Oviedo |
A | 42.07 | FHSAA 4A District 3 |
| 6 | Wekiva |
A | 42.43 | 2026 Dr. Baptiste Invitational hosted by West Orange High |
| 7 | University (Orlando) |
A | 43.60 | Lake Nona - Tammy Lafayette Relays 2025 |