| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Bradenton Christian |
A | Delgado | McArthur | Sanchez | Bailey | 48.42 | Saint Stephen's Falcon Invite 2026 |
| 2 | Southeast |
A | Johnson | Walker | Nawrot | Williams | 43.85 | Eddie Starling Memorial Invite |
| 3 | Booker |
A | Houston | Estridge | Sellers | Mobley | 43.17 | |
| 4 | Parrish |
A | Simeus | Michaud | Andrew | Byer | 42.76 | Larry Richardson Memorial Invite |
| 5 | Palmetto |
A | Delgado | Roberson | Scott | Everett | NT | |
| 6 | Bayshore |
A | Austin | Colston | Davis | Gibson | 43.25 | |
| 7 | Braden River |
A | Byrd | Dixon | Garvin | Stevens | 46.82 | Palmetto Freshman/Sophomore Ch |