| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note | 
|---|---|---|---|---|---|---|---|---|
| 3 | Lake Highland Prep | B | NeJame | Bissessar | Vogel | Bispo | NT | |
| 4 | Orlando Christian Prep | A | Gunn | NT | ||||
| 5 | Lake Mary | B | Brock-McEntire | Hollie | Williams | NT | 
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note | 
|---|---|---|---|---|---|---|---|---|
| 1 | Bishop Moore | A | Dunn | Nieves | Clermont | NT | ||
| 2 | Astronaut | A | Washington | McCray | Hamilton | Lamothe | NT | |
| 3 | Trinity Prep | A | Russell | Bodnar | Wilcox | Hatchette | NT | |
| 4 | Hagerty | A | Jacob | Jaime | Farwig | McGrath | 1:51.00 | |
| 5 | First Academy (Orlando) | A | Powelson | Hebert | Lozada Da Silva | 2:02.87 | Evans Trojans Relays | |
| 6 | Lake Highland Prep | A | Bacon | Vidrine | Simpson | Canine | NT | |
| 7 | Lake Mary | A | Iva | Bryant | Gaspar | Wood | NT |