| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Seabreeze |
A | 52.07 | Cocoa Tigers Relays Invitational |
| 2 | Bartram Trail |
A | 51.02 | 4th Annual North Florida/South Florida Challenge |
| 3 | Mandarin |
A | DNF | Mandarin Developmental Meet #2 (Approved Qualifier) |
| 4 | Fleming Island |
A | 50.64 | Creekside Friday Knight Invite |
| 5 | Flagler Palm Coast |
A | 51.32 | East River Invitational |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Bishop Snyder |
A | DNS | Bishop Kenny JV Meet |
| 3 | Matanzas |
A | 52.72 | ROYAL INVITATIONAL @ The First Academy 2025 |
| 4 | Fernandina Beach |
A | 58.89 | The Bulldog Classic Track and Field Meet |