| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 3 | Menendez | A | NT | |
| 4 | New Smyrna Beach | A | 53.70 | East Coast Classic |
| 5 | Lake Weir | A | 53.75 | FHSAA 3A Region 1 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Horizon | A | 52.98 | St. Cloud Invitational |
| 2 | Father Lopez | A | 52.30 | |
| 3 | Mainland | A | 51.95 | East Coast Classic |
| 4 | Eustis | A | 51.36 | FHSAA 2A Region 2 |
| 5 | Pine Ridge | A | 51.45 | FHSAA 2A Region 2 |
| 6 | Lake Minneola | A | 52.13 | Cypress Creek (Orlando) Pre-Season Meet |
| 7 | University (OC) | A | 52.63 | 52.63 Deltona HS Senior Night Meet |