| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Indian Rocks Christian |
A | NT | |
| 3 | Babcock | A | 5:04.84 | Saint Stephens Falcon Invite 2025 |
| 4 | Calvary Christian (PtStL) | A | 5:01.42 | Bulldog Invitational 2025 |
| 5 | Duval Charter Baymeadows |
A | DNS | Panther Invite at Ridgeview High School |
| 6 | Bradenton Christian |
A | DNS | Lakewood Ranch Invitational 2025 |
| 7 | Cambridge Christian |
A | NT |