| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Glenridge Middle School | A | Harris | Sanchez | Moore | King | 1:02.00 | |
| 2 | Arbor Ridge | A | Meyer | Williams | Hurlbert | Troyer | DNS | |
| 3 | Union Park Middle School | A | Anthony | Bullock | Ramos | Lee | 57.72 | |
| 4 | Maitland MS | A | Burns | Kiser | Jackson | Rushing | 54.80 | |
| 5 | OCPS ACE | A | Walker | Reeze | Willis | Johnson | 57.00 | |
| 6 | College Park Middle School | A | Buchanon | Previlus | Bowdery | Hughes | 58.00 | |
| 7 | Audubon Park School K-8 | A | Downs | Goodin | Sheppard | Timmons | 1:00.37 |