| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Okeechobee |
B | Kelly | Bloom | Maitland | Holder | 1:04.48 | Avon Park Red Devil Invitational |
| 4 | DeSoto County |
A | King | Brown | Johnson | Johnson | 55.34 | Avon Park Invitational |
| 5 | Okeechobee |
A | Pineda | Ridley | Bowman | Fonseca | 1:04.48 | Avon Park Red Devil Invitational |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Hardee |
A | Jackson | Silva | Pierre | Nunez | 53.44 | Bob English Invitational 2026 |
| 3 | Sebring |
A | Gray | Greene | Wellington | Madison | 52.35 | Avon Park Red Devil Invitational |
| 4 | Avon Park |
B | Glabach | Tate | Montiel | McMillan | 52.26 | Avon Park Red Devil Invitational |
| 5 | Avon Park |
A | Davi | Watt | Destino | Johnson | 52.26 | Avon Park Red Devil Invitational |
| 6 | LaBelle |
A | Deas | Williams | Sandoval | Jackson | 52.65 | CSN Invitational Powered by UnderArmour (Qualifier) |
| 7 | Moore Haven |
A | Herrera | Myers | Hughes | Faison | 53.54 | Jensen Beach Invitational 2026 |