| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | New Dimensions |
A | Khan | Saez-Pacheco | Vlahos | Rodriguez | DNS | |
| 4 | Victory Christian (LL) |
A | Gray | Gray | Scott | Cunningham | 45.00 | |
| 5 | Cornerstone Charter |
A | Borell | Vickers | Marsingill | Vazques-Marquez | 49.20 | Pioneer Invitational |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | IMG Academy |
A | Furishima | Shimizu | Ewing | Reeves | DNS | |
| 3 | Holy Trinity |
A | McMillan | Gillin | Sullivan | McRoy | 43.80 | |
| 4 | Foundation Academy |
A | McFarland | Rogers | Jean | Bronaugh | 41.45 | FHSAA Class 1A Track & Field State Championships |
| 5 | Edgewater |
A | Paul | Carter | Perry | Howard | 42.25 | |
| 6 | Hagerty |
A | Henson | Rhodes | Hatfield | Howell | 43.86 | |
| 7 | First Academy (Orlando) |
A | Wilson | Obatimilehin | Mason | Gardner | 44.83 |