| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Moore Haven |
A | Bowers | Ash | Kelly | Santana | NT | |
| 4 | Avon Park |
A | Glabach | Chamberlain | Virkler | Delgado | 52.48 | Tenoroc High School Invitational at SEU |
| 5 | Lake Placid |
A | Wu | Pattie | Claudio | Street | NT | |
| 6 | Okeechobee |
A | Holder | Bloom | Maitland | Bowman | NT |