| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed |
|---|---|---|---|---|---|---|---|
| 1 | Lake Weir | A | Burt, Jamarion | King, Zian | Dillard, Brian | Henry, Zadeen | 47.30 |
| 2 | Master's Academy (Oviedo) | A | Gillis, Josh | Darling, Blaize | Simpson, Evan | Gibbs, Terrance | 46.55 |
| 3 | Ridge Community | A | Centeno Serrano, Rubin | Del Rosario, Damian | Clark, Emanuel | Berry, Josh | 45.97 |
| 4 | Windermere Prep | A | Noel, Joel | Daniels, Seth | Allen, Najji | Martin, Wade | 44.00 |
| 5 | Cypress Creek | A | Arroyo, Emmanuel | Vertilus, Neil | Mcglocking, Caleb | Ughulu, Leslie | 45.55 |
| 6 | Villages | A | Martinez, Jonathan | Bryant, Brent | Ogerio, Arnold | Miller, Joseph | DQ |
| 7 | Oviedo | A | Howell, Jonathan | Almonte, Isaias | Johnson, Trendarius | Moore, Kashif | 46.59 |
| 8 | Leesburg | A | Coffee, Eric | Adams, Damarus | Jackson, Nicolas | Howard, Christian | 47.84 |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed |
|---|---|---|---|---|---|---|---|
| 2 | Montverde Academy | A | Fields , Tyler | Grimaud , Armani | Vaughn, Gregory | Baptist , Lincoln | DQ |
| 3 | Mount Dora Christian | A | Allen, Tyler | Bryant, Isaiah | Bryant, Rodney | Smith, Omari J | NT |
| 4 | Citrus | A | Rodriguez, Leonel | Devose, Erskine | Shamblin, Antonio | Weems, Terry | DNS |
| 5 | Foundation Academy | A | Wall, Weston | Andral-Colon, Jaden | Higgins, Bradley | Tallis, Parker | 52.23 |
| 6 | St. Francis | A | Cintron, Diego | Waiters, Zachary | Sabo, Chris | Hebert, Jarius | NT |
| 7 | Freedom (Orlando) | A | NT |