| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Hagerty | A | NT | |||||
| 2 | Master's Academy (Oviedo) | A | Batarseh | Joseph | Boktor | Bowles | 50.50 | FHSAA 2A Region 4 and FHSAA 1A Region 3 |
| 3 | East Ridge | A | Udom | Concepcion | Akomeah | Mickens | 49.62 | Windermere Meet of Champions 2021 |
| 4 | Olympia | A | Rodgers | Roundtree | Senat | Massey | 48.08 | Florida Relays |
| 5 | Lake Minneola | A | Gibson | Smith | Smith | Williams | 48.64 | Florida Relays |
| 6 | Lyman | A | Simpson | Lowe | Perez | Maze | DNS | FHSAA Class 4A-District 2 |
| 7 | Lake Brantley | A | Eccles | McMinn | Gomez | Brooks | 51.30 | FHSAA 4A District 2 |