| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Foundation Christian |
A | DNS | |
| 2 | Babcock | A | 4:05.31 | Palmetto Ridge Bear Classic |
| 3 | Duval Charter Baymeadows |
A | DNS | Tocoi Creek Toro Showdown 2025 (Approved Qualifier) |
| 4 | Indian Rocks Christian |
A | 3:35.64 | Calvary Christian Warrior Invitational |
| 5 | Cambridge Christian |
A | 3:37.63 | Bruce Calhoun Invitational |
| 6 | Classical Academy (Sarasota) |
A | 4:03.80 | Bobcat Relays |
| 7 | Calvary Christian (PtStL) | A | 4:11.68 | Bulldog Invitational 2025 |