| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Lake Region |
A | Alvarez | Penley | Lucas | Campbell | DNS | Lake Region Sprint Meet 2026 |
| 2 | Frostproof |
A | Andrews | Ramirez | Aquirre Sanchez | Shields | 3:48.24 | Lake Region HS Distance Carnival 2026 |
| 3 | Bartow |
A | Jenking | Callins | Robinson | Brinson | 3:42.28 | Blue Devil Invitational (Qualifier) |
| 4 | Lake Wales |
A | Daniels | Walker | Pugh | Daniels | DNS | Berkeley Track and Field Invitational |
| 5 | Ridge Community |
A | Palmer | Yanez-Domingu | Davis | Patino | 3:37.07 | Lake Region Sprint Meet 2026 |
| 6 | Discovery |
A | Fernandez | Williams | Sewnarine | Maisonet | 3:43.86 | Winter Haven Classic |
| 7 | Haines City |
A | Bordes | Andrews | Peters | Brooks | 3:49.69 | Bob English Invitational 2026 |
| 8 | Mulberry |
A | Jones | Morales | OSteen | Smith | 4:07.07 | Lake Region Sprint Meet 2026 |