| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Cornerstone Charter |
A | Cumbal | Marsingill | Mendez | Vazques-Marquez | 47.41 | ROYAL INVITATIONAL @ The First Academy 2025 |
| 2 | Freedom (Orlando) |
A | Rojas | Padrino | Liu | Draper | 44.81 | Tiger Invitational |
| 3 | Oak Ridge |
A | Nkuitabong | Suffrant | Oracius | Altidor | 43.35 | ROYAL INVITATIONAL @ The First Academy 2025 |
| 4 | Evans |
A | Bryant | Hardy | Pompilus | Gumbs | DNS | Bob Hayes High School Invitational |
| 5 | Dr. Phillips |
A | Horne | Scott | Fortilien | Pierre | 43.10 | Charlie Harris Relay |
| 6 | Edgewater |
A | Martin | Jenkins-Lee | Perry | Sterling | 43.60 | Tiger Invitational |
| 7 | Innovation_Orlando | A | Iskander | Soto | Espinal Ortega | Mercado-White | 45.40 | Ocoee Invitational |
| 8 | Colonial |
A | Gonzalez | Daniels | Garcia-Nazario | Jackson | DNS |