| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Colonial |
A | Gonzalez | Scott | Garcia-Nazario | Jackson | NT | |
| 3 | Foundation Academy |
A | Camacho | McMullen | Mercado | Waldon | 47.00 | |
| 4 | Boone |
A | Noelsaint | Davis | Lance | Hardy | 44.64 | FHSAA 4A District 9 |
| 5 | Innovation_Orlando | A | Dendy | Kest | Harris | Moore | 45.40 | Ocoee Invitational |
| 6 | Master's Academy (Oviedo) |
A | Evans | Jenkins | Martin | Chadwick | 52.00 | |
| 7 | Horizon |
A | Foga | Rector | Landgraf da Fonseca | Russo Braz | DNS |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Ocoee |
A | Hales lll | Goins | Archer | Paul | 43.62 | |
| 2 | Windermere |
A | Todd | Miesak | George | Bronaugh | 43.00 | |
| 3 | Dr. Phillips |
A | Pierre | Greaves | Mincey | Matthews | 42.95 | 42.95 Kowboy Invitational |
| 4 | Edgewater |
A | Martin | Sterling | Johnson | Jenkins-Lee | 41.55 | FHSAA 3A Region 2 |
| 5 | Oviedo |
A | Galarza | Washington | Slivinski | Nixon | 42.53 | 42.53 Charlie Harris Relays |
| 6 | West Orange |
A | Brown | Carson | Glover | Dillard | 42.96 | 42.96 West Orange - Dr. Baptiste Invitational 2024 |
| 7 | Wekiva |
A | Dixon | Saint George | Manley | Garvin | 43.26 | 43.26 Charlie Harris Relays |
| 8 | Apopka |
A | Gantz | Williams | Gibbs | Lambert | 44.50 |