| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Mulberry |
A | Jones | Campbell | Morales | OSteen | 4:07.07 | Lake Region Sprint Meet 2026 |
| 3 | DeSoto County |
A | Tanner | Hernandez | Shine | Johnson | 3:51.69 | Dunbar Tiger Classic Invite (Approved Qualifier) |
| 4 | Sebring |
A | Volvaire | Bostick | Turner | Hamilton | 3:35.39 | Bob English Invitational 2026 |
| 5 | Avon Park |
A | Jones | Roberts | Owens | Payne | 3:39.02 | Avon Park Red Devil Invitational |
| 6 | Lake Placid |
A | Vega | Leon | Falls | Abuelouf | DNS | Avon Park Red Devil Invitational |