| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | South Lake |
A | 45.52 | Dr. Phillips Panther Invitational |
| 3 | Gateway (Fort Myers) |
A | 44.70 | 10th Annual Loretta Purish Invitational |
| 4 | Hardee |
A | 43.65 | FHSAA 2A Region 3 |
| 5 | Lake Placid |
A | 44.46 | FHSAA 2A District 11 |
| 6 | LaBelle |
A | DNS | Bishop Verot - Viking Elite Classic (Regional Qualifier) |
| 7 | DeSoto County |
A | 45.86 | Sebring Relays 2024 |