| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Middleton |
A | Stanford | Cason | Hardy | Lawrence | 45.21 | 45.21 2025 Hillsborough County National Conference Championship |
| 4 | Chamberlain |
A | Wright | DeLoatch | Ford | Winns | 42.55 | FHSAA 2A District 10 |
| 5 | Tampa Catholic |
A | Billingsley | Weigert | Monette | Jones | 43.85 | Tampa Bay Private School Track and Field Championships |