| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Roosevelt_Academy | A | NT | |
| 3 | Okeechobee |
A | 53.04 | MCHS Sarge Invitational 2025 |
| 4 | Lake Placid |
A | 54.69 | Avon Park Red Devil Invitational |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | LaBelle |
A | DNS | FHSAA 3A District 11 |
| 2 | Sebring |
A | 51.93 | Lake Placid Invitational 2026 |
| 3 | Frostproof |
A | 51.35 | FHSAA 2A District 9 |
| 4 | Hardee |
A | 51.62 | FHSAA 2A Region 3 |
| 5 | DeSoto County |
A | 52.06 | Sebring Meet #2 2025 |
| 6 | Avon Park |
A | 52.26 | Avon Park Red Devil Invitational |