| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed |
|---|---|---|---|---|---|---|---|
| 1 | Mitchell |
A | Varona | Mirable | Blasewitz | Kraujalis | NT |
| 2 | Springstead |
A | Cordero | Ihejirika | Grant | Stanina | 3:50.00 |
| 3 | Cypress Creek (Wesley Chapel) |
A | Clifton | Knowles | Yuengling | Ibironke | 3:47.53 |
| 4 | Land O'Lakes |
A | Faria | Vazquez | Perez | Agbagu | 3:28.46 |
| 5 | Wiregrass Ranch |
A | Dimarco | Mathurin | Stewart | Harrod | 3:34.52 |
| 6 | Zephyrhills |
A | Stone | Brown | Keating | Warren | NT |
| 7 | Angeline Academy | A | Liles | Doobay | Petrovich | Lowe | 3:56.28 |
| 8 | Gulf |
A | Porter-Jackson | Coleman | Brown | Carpenter | NT |