| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Clewiston |
A | Magana | Graham | Love | Coring | 4:05.11 | Jensen Beach Invitational 2026 |
| 2 | DeSoto County |
A | Grant | Shine | Hernandez | Johnson | 3:51.69 | Dunbar Tiger Classic Invite (Approved Qualifier) |
| 3 | Avon Park |
B | Burgess Jr. | Severo | Washington | Washington | DNS | Avon Park Red Devil Invitational |
| 4 | Sebring |
A | Volvaire | Bostick | Turner | Hamilton | 3:34.71 | Avon Park Invitational |
| 5 | Avon Park |
A | Jones | Payne | Roberts | Owens | 3:39.02 | Avon Park Red Devil Invitational |
| 6 | Hardee |
A | Dick | Bryant | Guevara | Francois | 3:49.61 | Avon Park Red Devil Invitational |
| 7 | Lake Placid |
A | Melgar | Leon | Roblero-Flores | Abuelouf | 3:55.68 | Avon Park Red Devil Invitational |
| 8 | LaBelle |
A | Williams | Simmons | Maldonado | Thomas | NT |