| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Eustis | A | NT | |
| 3 | Horizon | A | NT | |
| 4 | North Marion | A | DQ | |
| 5 | Menendez | A | NT |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | University (OC) | A | 46.22 | 5-Star Conference Meet |
| 2 | Lake Weir | A | 45.32 | Trinity Catholic Tuesday Invitational |
| 3 | Mainland | A | 43.45 | East Coast Classic |
| 4 | Lake Minneola | A | 42.16 | Florida Relays |
| 5 | New Smyrna Beach | A | 43.42 | FHSAA 3A District 6 |
| 6 | DeLand | A | 44.18 | 5-Star Conference Meet |
| 7 | Father Lopez | A | 45.36 | East Coast Classic |