| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Calvary Christian (PtStL) | A | 58.94 | Jensen Beach Invitational 2025 |
| 3 | Duval Charter Baymeadows |
A | DNS | Running of the Jackets |
| 4 | Babcock | A | 53.75 | Saint Stephens Falcon Invite 2025 |
| 5 | Bradenton Christian |
A | 54.47 | John Tasopolos Memorial Invitational |
| 6 | Cambridge Christian |
A | 56.51 | Calvary Christian Warrior Invitational |
| 7 | Indian Rocks Christian |
A | 59.69 | Shorecrest Charger Invitational |