| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Lake Worth |
A | 50.32 | Royal Palm Beach Invitational 2025 |
| 2 | New Smyrna Beach |
A | 50.17 | FHSAA 3A Region 2 |
| 3 | Seminole (Sanford) |
A | 48.38 | FHSAA State Track & Field Championships (4A) |
| 4 | Wekiva |
A | 46.10 | FHSAA State Track & Field Championships (4A) |
| 5 | Wekiva |
B | 46.10 | FHSAA State Track & Field Championships (4A) |
| 6 | Mainland |
A | 49.01 | FHSAA 3A Region 2 |
| 7 | New Smyrna Beach |
B | 50.17 | FHSAA 3A Region 2 |
| 8 | Winter Park |
A | 50.72 | FHSAA 4A Region 1 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Atlantic (PO) |
A | 56.44 | Deltona HS - Wolves Tuneup 2026 |
| 3 | Orlando Christian Prep |
A | 53.86 | Mount Dora Christian Invitational Two |
| 4 | Menendez |
A | 51.08 | Royal Invitational @ The First Academy 2026 |
| 5 | Spruce Creek |
A | 51.55 | 5-Star Conference |
| 6 | University (OC) |
A | 55.35 | Sanford-Seminole Open 2025 |
| 7 | Atlantic (PO) |
B | 56.44 | Deltona HS - Wolves Tuneup 2026 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 3 | Menendez |
B | NT | |
| 4 | Halifax Academy |
A | 1:03.47 | 2026 Raines Developmental Meet #3 |
| 5 | Orlando Christian Prep |
B | NT |