| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Dunnellon |
A | Hytovick | De La Rosa | Crawford | Walker | 53.03 | Wildwood Open |
| 4 | Wildwood |
A | Solomon | Dempsey | Williams | Parker | 48.72 | FHSAA 1A Region 2 |
| 5 | Lake Minneola |
A | Green | Herrera | McGlynn | Rose | 52.59 | FHSAA 4A Region 1 |
| 6 | Citrus |
A | Bailey | Budd | Taylor | Shiano | 55.64 | Doug Patton Invitational |