| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | OCPS ACE | A | White | Willis | Bradley | McKee | 52.00 | |
| 3 | Union Park Middle School | A | Collins | Cintron | Ortiz | Horne | 51.00 | |
| 4 | Maitland MS | A | Weeks | Wallace | Ross | Glomski | 49.70 | |
| 5 | Arbor Ridge | A | Peguero | Leon | Rivera | Ortiz | 50.81 | |
| 6 | Audubon Park School K-8 | A | Hall | McAuliffe | Faulkner | Aiosa | 51.04 | |
| 7 | Glenridge Middle School | A | Wilson | Johnson | Altman | Dickson | 54.10 | |
| 8 | College Park Middle School | A | NT |