| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Orangewood Christian |
A | Bulger | Kaiser | Bowie | Toadvine | NT | |
| 2 | Tohopekaliga |
A | Oliver | Hall | Veras | Marquez | 44.84 | 44.84 St. Cloud Track Invitational |
| 3 | Lake Highland Prep |
A | Francis | Safi | Torres | Rivera | 43.50 | |
| 4 | Winter Park |
A | Leonard | Gardner | Washington | Diaz | 42.47 | 42.47 Charlie Harris Relays |
| 5 | Oviedo |
A | Slivinski | Hernandez | Nixon | Galarza | 42.02 | FHSAA 4A Region 1 |
| 6 | Jones |
A | Nichols | Smith | Wilson | Robbins | 42.44 | |
| 7 | Mainland |
A | Estes | Edwards | Hill | Shine | DNF | |
| 8 | University (Orlando) |
A | Williams | Agard | McGinnis | Sylvester | 44.30 | Metro East Championship 2024 |
| 9 | Trinity Prep |
A | Artman | Lou | Wang | Davis-Rocha | DNF |