| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | St. Petersburg |
A | Gainer | Manning | Uhrmacher | Gibson | 43.99 | George Steinbrenner Invitational |
| 3 | Clearwater |
A | Gooden | Smith | Benjamin | Chambers | 44.07 | Dunedin High Falcon Relays |
| 4 | Seminole |
A | Abbot | Vaughan | Jarvis | Currie | 44.58 | 44.58 John Tasopolos Memorial Invitational |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Gibbs |
A | Delgado | Alexis | Roberson | Wall | 41.85 | FHSAA 2A Region 3 |
| 3 | Boca Ciega |
A | Williams | Ward | Gray | Edwards | 41.29 | FHSAA State Track & Field Championships (2A/Para-Athlete) |
| 4 | Largo |
A | Cooper | Whitaker | Rackards | Smith | 41.46 | 41.46 Mustang Invitational |
| 5 | Lakewood |
A | Dent | Davis | Nixon | Webb | 42.59 | Larry Richardson Memorial Invite |