| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | HUNTERS CREEK M.S. | A | DE OLIVEIRA MACHADO | HOFFMAN | REINOSA DE JESUS | SALLES | 4:38.00 | |
| 2 | Luminary Middle School | A | Liboy | Austin | Crellin | Sardon | 4:21.00 | |
| 3 | Lake Nona Middle School | A | Salas Colon | Liriano | Chan | Vega | 4:16.35 | |
| 4 | South Creek Middle School | A | McCall | Ramirez | Villasana | Josue | 4:15.00 | |
| 5 | Walker Middle School | A | Paul | Lucas-Martinez | Williams | Henry | 4:16.00 | |
| 6 | Innovation Middle School | A | Phillips | Mckenzie | Paiva | Geers | 4:17.00 | |
| 7 | Meadow Woods Middle School | A | Portal | Spann | Banks | Lima | 4:30.00 | |
| 8 | Freedom Middle School | A | Henderson | Thompson | Gonzalez | Thornton | 5:01.78 |