| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Lake Highland Prep |
A | Canine | Bacon | Simpson | Vidrine | NT | |
| 2 | Winter Park |
A | Pierre | Byrd | Davis | Bernhardt | 57.03 | 57.03 East River Developmental Meet |
| 3 | Tohopekaliga |
A | Pagan | Martinez | Diaz Monge | Francois | 52.15 | Orange Belt Conference Championships |
| 4 | University (Orlando) |
A | Morris | Vassor | Neance | Gilmer | DNF | Metro East Championship 2024 |
| 5 | Lake Brantley |
A | Ary | Edwards | Berehausky | Nwaogwugwu | 49.06 | |
| 6 | Jones |
A | Ocasio | Williams | Sheffield | Heard | 49.47 | 49.47 Ocoee Relays |
| 7 | Mainland |
A | Watlington | Key | Joseph | Williams | 51.49 | Spruce Creek Last Chance Meet (FHSAA Sanctioned Meet) |
| 8 | Trinity Prep |
A | Burgos | Moore | Pelin | Yared | 52.64 | 52.64 Trinity Prep Relays 2023 |
| 9 | Oviedo |
A | Vecchiarelli | Wahl | Wahl | Keke | NT |