| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Braden River | A | Saint-jean | Bolduc | Arrington-Bey | Dionela | 51.36 | Charlotte Invitational |
| 2 | Venice | A | Ayette | Ayette | Legare | James | 49.91 | FHSAA 3A Region 3 |
| 3 | Manatee | A | Gibbs | Wiggins | Williams | Wiggins | 49.03 | FHSAA 4A Region 2 and FHSAA 3A Region 2 |
| 4 | North Port | A | Jean | Jefferson | Coyle | Mask | 49.88 | FHSAA 4A Region 2 and FHSAA 3A Region 2 |
| 5 | Bishop Verot | A | Gulati | Lewis | Taglieri | Herrera | 50.14 | FHSAA 2A Region 3 and FHSAA 1A Region 2 |
| 6 | Booker | A | Ash | Storr | Thomas | Arrington | 51.39 | FHSAA 2A Region 3 and FHSAA 1A Region 2 |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Riverview (Sarasota) | A | Shriver | Manitz | Fegan | Hamer | 52.95 | Sarasota Riverview Ram Invite |
| 3 | Sarasota | A | Ratner | Oliver | Scrimale | Schwied | 51.50 | |
| 4 | Cardinal Mooney | A | Via-Reque | Rogers | Harell | Smith | DQ | FHSAA 2A Region 3 and FHSAA 1A Region 2 |