| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Cornerstone Charter |
A | Cumbal | Cumbal | Gough | Harrington | DNS | |
| 2 | Freedom (Orlando) |
A | Rijos | Thompson | Soyez | Guevara | 3:34.53 | Tiger Invitational |
| 3 | Edgewater |
A | Biglow | Collin | Moore | Houston | DNS | Tiger Invitational |
| 4 | Oak Ridge |
A | Hall | Collins | Thadal | Docteur | 3:28.05 | Blue Devil Invitational (Regio |
| 5 | Evans |
A | Denmak | Gumbs | Rivers | Clarke | 3:28.54 | Wolverine Relays |
| 6 | Dr. Phillips |
A | Scott | Williams | Davis | Mescharles | DNS | Bob English Invitational 2025 |
| 7 | Innovation_Orlando | A | Dendy | Mercado-White | Davila | Kest | 3:44.16 | Harmony Invitational |
| 8 | Colonial |
A | Martinez | Garcia-Nazario | Castaneda | Jackson | DNS |