| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Crooms | A | FRANCIS | Gallimore | Barker | Rhodes | DNS | |
| 3 | Hagerty | A | Trapaga Morejon | Jaime | Farwig | Nowak | 52.00 | |
| 4 | Lake Mary | A | Harris | Negron | Singletary | Smith | 49.36 | FHSAA 4A Region 1 |
| 5 | Winter Springs | A | Allo | Schickling | Gayman | 51.36 | ||
| 6 | Oviedo | A | Wahl | Harshbarger | Conzemius | Vecchiarelli | NT | |
| 7 | Lake Brantley | A | Ary | Berehausky | Edwards | Pierre Louis | NT |