| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Lake Wales |
A | St. Fleur | Parades-Pinerez | Turner | Green | DNS | Berkeley Track and Field Invitational |
| 3 | Haines City |
A | Bell | Tarver | Wroy | Page | 4:25.76 | TFRRS 02/28/26 - 4:15 |
| 4 | Bartow |
A | Upthegrove | Moore | Harris | Shahan | 4:16.67 | Blue Devil Invitational (Qualifier) |
| 5 | Discovery |
A | Buckle | Blair | Buckle | Buckle | 4:24.68 | Winter Haven Classic |
| 6 | Ridge Community |
A | Jacinthe | Gleason | Vidal | Rivera | 4:32.09 | Tenoroc High School Invitational at SEU |
| 7 | Frostproof |
A | Jones | Tibado | Butler | Ruttan | NT |