| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Mulberry |
A | Cobbett | Townsend | OSteen | Nichols | 46.02 | Lake Region Sprint Meet 2026 |
| 3 | DeSoto County |
A | Wilkinson | Johnson | Johnson | Hamm | 44.79 | Pirate Booty Invitational- Approved Qualifier |
| 4 | Sebring |
A | Brown | Austin | Walker | Hamilton | 43.27 | Bob English Invitational 2026 |
| 5 | Avon Park |
A | Owens | Jones | Jones | Owens | 43.88 | Avon Park Red Devil Invitational |
| 6 | Vanguard (LW) |
A | Khan | Seymour | Cartright | Lacossade | 45.90 | SFCHS Sprint & Hurdle Meet |
| 7 | Lake Placid |
A | Falls | Corby | Patterson | Abuelouf | 48.49 | Avon Park Red Devil Invitational |