| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Jefferson |
A | Molina | Carwell | Beauford | Hayes | 3:50.28 | Leto March 25th Track 2025 |
| 2 | Jesuit (Tampa) |
C | Richey | Jimenez | Smith | Pierre | 3:35.00 | |
| 3 | Tampa Catholic |
A | House | Jones | Stokes | Weigert | 3:25.72 | FHSAA State Track & Field Championships (2A/Para-Athlete) |
| 4 | Jesuit (Tampa) |
A | Acosta | Jackson | Rice | Ohlson | 3:33.14 | FHSAA 3A District 9 |
| 5 | Jesuit (Tampa) |
B | Lett | Olaleye | Depass | McGovern | 3:50.00 |