| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Out-of-Door |
C | NT | |
| 3 | Out-of-Door |
A | 55.77 | 55.77 Sarasota County Track and Field Championship |
| 4 | Bradenton Christian |
A | 53.63 | 53.63 ODA Thunder Classic 2025 |
| 5 | Babcock | A | 53.75 | Saint Stephens Falcon Invite 2025 |
| 6 | Out-of-Door |
B | NT |