| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | LaBelle |
A | 54.74 | Lake Placid Meet #2 |
| 2 | Gateway (Fort Myers) |
A | 53.89 | FHSAA 2A Region 3 |
| 3 | DeSoto County |
A | 53.79 | FHSAA 2A Region 3 |
| 4 | Immokalee |
A | 53.39 | FHSAA 3A Region 3 |
| 5 | Lake Placid |
A | 53.75 | Lake Placid Meet #2 |
| 6 | Hardee |
A | 53.79 | FHSAA 2A Region 3 |
| 7 | South Lake |
A | 53.95 | Winter Park - Brian Jaeger Elite Classic 2023 |