| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Tampa Prep |
A | Kunkel | Upshaw | Clark | Burke | 45.25 | Morgan Inaugural Track Meet |
| 4 | Newsome |
A | Martinez | Rabold | Jefferson | Keaton | DNF | 2026 Manuel Griffin Fraundorfer Relays |
| 5 | Spoto |
A | Jones | Bernard | Berry | Grider | DNF | FHSAA 3A District 9 |
| 6 | Strawberry Crest |
A | Hanson | Yeager | Martinez-Ramos | Brown | 47.50 |