| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed |
|---|---|---|---|---|---|---|---|
| 1 | Angeline Academy | A | Doobay | Petrovich | Januario | Ruhlig | DQ |
| 2 | Cypress Creek (Wesley Chapel) |
A | Pinson | Fountain | Knowles | Burkes | 44.47 |
| 3 | Zephyrhills |
A | Denson III | Miller | Pope | Pope | 43.78 |
| 4 | Gulf |
A | Porter-Jackson | Brown | Coleman | Carpenter | 42.92 |
| 5 | Wiregrass Ranch |
A | Drake | Austin | Stewart | Gonzalez | 43.35 |
| 6 | Mitchell |
A | Dunning | Connor | Beach | Harrigin | DQ |
| 7 | Springstead |
A | Benjamin | Debord | Marc | Moate | 45.50 |