| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Vanguard (Ocala) |
B | Powers | Sneed | Cox | Dixon | 48.21 | FHSAA 3A Region 2 |
| 3 | Mount Dora Christian |
A | Robles | Kudra | Filetti | Kudra | 49.83 | Mount Dora Christian Invitational Meet #2 |
| 4 | Vanguard (Ocala) |
A | Eady | Henderson | Laboo | Reason | 48.21 | FHSAA 3A Region 2 |
| 5 | Wildwood |
A | Corbin | Edwards | Williams | Colbert | 48.72 | FHSAA 1A Region 2 |
| 6 | South Lake |
A | Williams | Clements | Grilli | Clarke | 50.93 | Tiger Invitational |