| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Meadow Woods Middle School | A | Madero | Forehand | Zambrana | Sutherland | 53.50 | |
| 2 | Walker Middle School | A | Uribe | Williams | Jonson | Meneus | 51.99 | |
| 3 | Lake Nona Middle School | A | Vazquez | Hinds | Le | Corzo | 50.87 | |
| 4 | South Creek Middle School | A | Laureda | Santana-Bruno | Munoz | Torres | 49.77 | |
| 5 | Luminary Middle School | A | Pico | Boodoosingh | Alvarez | Bradbury | 50.49 | |
| 6 | Innovation Middle School | A | Freeman | Meckstroth | Nieves | Rajesh | 51.95 | |
| 7 | HUNTERS CREEK M.S. | A | KNIGHTON | VALEUS | DOS SANTOS DA SILVA | LANDAEZ | 52.06 | |
| 8 | Freedom Middle School | A | Ceneus | Mujica | Allen | Lopez | NT |