| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Master's Academy (Oviedo) | A | Wilbanks | Boivin | Brommer | Wallace | NT | |
| 4 | Hagerty | A | Sattler | Assenmacher | Hobbs | Douglas | 44.36 | Trinity Prep Invitational 2020 |
| 5 | Lyman | A | Plumber | Washington | Anderson | Palmer | DNS | Seminole Athletic Conference Championships 2019 |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Wekiva | A | Wilson | Clerisier | Joseph | Davidson | 42.78 | Kowboy Invitational 2019 |
| 3 | Olympia | A | Aliu-otokiti | Aliu-otokiti | Sullivan | Robinson | 42.29 | 2019 FHSAA State Track & Field Championships |
| 4 | Edgewater | A | Collins | Bass | Wallace | Sanders | 41.86 | FHSAA 3A Region 2 |
| 5 | East Ridge | A | Grafals | Orti | Vacca | Watts | 42.20 | Bob English Relays 2020 |
| 6 | Lake Brantley | A | Johansen | Spragg | Pierce | Silva | 42.44 | FHSAA 4A Region 1 |
| 7 | Lake Minneola | A | Thomas | Rogers | Carman | Stump | 43.40 | FHSAA Class 4A-District 3 |