| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Vanguard (Ocala) |
B | West | Cotman | Cox | Joseph | 4:22.08 | 2025 MAC Track and Field Championships |
| 3 | South Lake |
B | Caurthens-Lowe | Curry | Johnston | Florence | 4:20.25 | |
| 4 | Wildwood |
A | Williams | Colbert | Rollerson | Rivenberg | 4:08.51 | FHSAA 1A Region 2 |
| 5 | South Lake |
A | Clarke | Baldwin | Miller | Wong | NT | FHSAA 4A District 4 |
| 6 | Vanguard (Ocala) |
A | Osbourne | Sneed | Wilkerson | Powers | 4:22.08 | 2025 MAC Track and Field Championships |