| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | University (Orlando) |
B | Clarke | Curry | Duncan | Grullon | DNS | |
| 4 | Lake Nona |
B | Stokes | Manning | Seay | Scott | NT | |
| 5 | Innovation_Orlando | B | Delarosa | Hubbard | Altidor Jr | Rosado | NT | |
| 6 | First Academy (Orlando) |
B | Ivsic | Robinson | Hester | Powell | NT | |
| 7 | Windermere |
B | Derrick | Mosby | Collins | Lewis | NT |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Colonial |
A | Mintz | Santos | Pierre Louis | Thompson | NT | |
| 2 | Lake Nona |
A | Stokes | Lafayette | Henderson | Jones | NT | |
| 3 | University (Orlando) |
A | Rodriguez | Fenton | Lionel | Williams | NT | |
| 4 | Windermere |
A | Johnston | Banville | Rivas | Hay | 1:30.01 | |
| 5 | Tohopekaliga |
A | Lee | Weck | Marquez | Roman | NT | |
| 6 | First Academy (Orlando) |
A | Obatimilehin | Gardner | Jackson | Hickson | NT | |
| 7 | Innovation_Orlando | A | Dendy | Moore | Harris | Davila | NT |