| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Matanzas |
A | 4:17.41 | Lake Mary Invitational 2025 |
| 2 | Flagler Palm Coast |
A | 4:13.73 | Creekside Friday Knight Invite |
| 3 | Fleming Island |
A | 4:18.11 | 4th Annual North Florida/South Florida Challenge |
| 4 | Bartram Trail |
A | 4:12.59 | 2025 Danny Brown Invitational |
| 5 | Seabreeze |
A | 4:58.78 | 11th Annual Craig Speziale Memorial Track & Field Invite |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Bishop Snyder |
A | DNS | Bishop Snyder JV Meet #1 |
| 3 | Mandarin |
A | DNS | Tocoi Creek Toro Showdown 2025 (Approved Qualifier) |
| 5 | Orange Park |
A | DNS | 11th Annual Craig Speziale Memorial Track & Field Invite |