| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | East River |
A | 43.10 | FHSAA 4A District 3 |
| 2 | Wekiva |
B | 42.17 | Mustang Relays |
| 3 | Seminole (Sanford) |
A | 42.03 | 18th Annual George Janan Spruce Creek Invitational |
| 4 | Winter Park |
A | 41.94 | FHSAA 4A Region 1 |
| 5 | Spruce Creek |
A | 42.00 | |
| 6 | Wekiva |
A | 42.17 | Mustang Relays |
| 7 | Mainland |
A | 42.35 | 5-Star Conference |
| 8 | Menendez |
A | 43.32 | Embry-Riddle Running Elements Classic High School |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Atlantic (PO) |
B | 44.56 | Embry-Riddle Running Elements Classic High School |
| 2 | New Smyrna Beach |
A | 44.20 | 18th Annual George Janan Spruce Creek Invitational |
| 3 | Orlando Christian Prep |
B | 43.94 | Winter Park - Brian Jaeger Elite Classic 2025 |
| 4 | Deltona |
A | 43.71 | Deltona HS - Wolves Tuneup 2026 |
| 5 | Flagler Palm Coast |
A | 43.88 | |
| 6 | Orlando Christian Prep |
A | 43.94 | Winter Park - Brian Jaeger Elite Classic 2025 |
| 7 | New Smyrna Beach |
B | 44.20 | 18th Annual George Janan Spruce Creek Invitational |
| 8 | Atlantic (PO) |
A | 44.56 | Embry-Riddle Running Elements Classic High School |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Menendez |
B | NT | |
| 3 | Seabreeze |
B | 44.87 | 12th Annual Craig Speziale Memorial Invitational |
| 4 | West Port |
A | 44.75 | 2025 Mandarin Classic (Approved Qualifier) |
| 5 | Seabreeze |
A | 44.87 | 12th Annual Craig Speziale Memorial Invitational |
| 6 | University (OC) |
A | 45.75 | 5-Star Conference |
| 7 | Halifax Academy |
A | NT |