| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed |
|---|---|---|---|---|---|---|---|
| 1 | Oviedo | A | Wahl | Vecchiarelli | Harshbarger | Wahl | DNS |
| 2 | Winter Springs | A | Brown | Huet | Jimenez | Giambalvo | 51.36 |
| 3 | Lake Howell | A | Lowe | Golson | Dudley | Stokes | 49.81 |
| 4 | Lake Mary | A | Jiles | Reese | Brooks | Iva | 49.36 |
| 5 | Seminole (Sanford) | A | Soremi | Soremi | Hicks | Allen | 49.50 |
| 6 | Lake Brantley | A | Ary | Berehausky | Edwards | Nwaogwugwu | 51.01 |
| 7 | Hagerty | A | Jacob | Jaime | Farwig | Morley | 52.02 |
| 8 | Crooms | A | NT |