| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Seabreeze |
A | DNS | Volusia/Flagler Fresh/ Sophomore Meet |
| 2 | Tocoi Creek |
A | DNS | Tocoi Creek Toro Showdown 2025 (Approved Qualifier) |
| 3 | Ponte Vedra |
A | DNS | Creekside Friday Knight Invite |
| 4 | Matanzas |
A | 44.34 | 11th Annual Craig Speziale Memorial Track & Field Invite |
| 5 | Fernandina Beach |
A | DNS | 11th Annual Craig Speziale Memorial Track & Field Invite |
| 6 | Bishop Snyder |
A | 47.19 | Cardinal Invitational - Bishop Snyder |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Orange Park |
A | 43.58 | Sam Burley Hall of Fame Invitational |
| 2 | Fleming Island |
A | 43.20 | 11th Annual Craig Speziale Memorial Track & Field Invite |
| 3 | Flagler Palm Coast |
A | 42.54 | 4th Annual North Florida/South Florida Challenge |
| 4 | Bolles |
A | DNS | The Bulldog Classic Track and Field Meet |
| 5 | Mandarin |
A | 43.28 | Mandarin Developmental Meet #2 (Approved Qualifier) |
| 6 | Bartram Trail |
A | 44.20 | 2025 Danny Brown Invitational |