| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Avon Park |
A | Watkins | McGuire | Hurd | Severo | 3:56.65 | Frostproof - The Ridge Open 2025 |
| 4 | Hardee |
A | Thompson | Moran | Brunson | Myrick | 3:42.26 | Larry Richardson Memorial Invitational |
| 5 | DeSoto County |
A | Morgan | Barrare | Tice | Shine | 3:46.79 | Avon Park Meet #1 2025 |
| 6 | Lake Placid |
A | Anderson | Walton | Washington | Charite | NT |