| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Bartram Trail |
A | 3:34.27 | 11th Annual Craig Speziale Memorial Track & Field Invite |
| 2 | Flagler Palm Coast |
A | DNS | 4th Annual North Florida/South Florida Challenge |
| 3 | Bolles |
A | DNS | The Bulldog Classic Track and Field Meet |
| 4 | Mandarin |
A | 3:24.96 | Tocoi Creek Toro Showdown 2025 (Approved Qualifier) |
| 5 | Orange Park |
A | 3:31.30 | Sam Burley Hall of Fame Invitational |
| 6 | Matanzas |
A | 3:34.42 | ROYAL INVITATIONAL @ The First Academy 2025 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Bishop Snyder |
A | 3:44.53 | 2025 Danny Brown Invitational |
| 3 | Tocoi Creek |
A | 3:35.39 | Tocoi Creek Toro Showdown 2025 (Approved Qualifier) |
| 4 | Fleming Island |
A | 3:39.28 | 21st Annual East Coast Classic |
| 5 | Seabreeze |
A | DNS | 12th Annual Flagler Pinnacle Pre-Season Classic |