| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Calvary Christian (PtStL) | A | 53.18 | Jensen Beach Invitational 2025 |
| 3 | Babcock | A | 48.35 | Saint Stephens Falcon Invite 2025 |
| 4 | Duval Charter Baymeadows |
A | DNS | Panther Invite at Ridgeview High School |
| 5 | Cambridge Christian |
A | 47.90 | Mustang Invitational |
| 6 | Foundation Christian |
A | DNS | 2025 Newsome Wolves Invitational |
| 7 | Indian Rocks Christian |
A | NT |