| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Out-of-Door |
B | NT | |
| 3 | Bradenton Christian |
A | 5:19.71 | 5:19.71 Lakewood Ranch Invitational 2025 |
| 4 | Out-of-Door |
A | 4:37.17 | Saint Stephens Falcon Invite 2025 |
| 5 | Babcock | A | 4:41.83 | Sunshine State Athletic Association Championship 2025 |
| 6 | Classical Academy (Sarasota) |
A | NT |