| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Horizon |
A | Astorino | Seidu | Wilson | Geyer | NT | |
| 4 | Windermere |
A | Brown | Gordon | Miller | Robinson | DNS | Ocoee Invitational (Meet is Full) |
| 5 | Innovation_Orlando | A | Roderick | Londoni | Fitzherbert | Behzad | 4:49.76 | Harmony Invitational |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Colonial |
A | Sukut | Smith | Arroyo | Rodriguez | 4:21.03 | 4:21.03 Metro East Championship 2021 |
| 2 | Oviedo |
A | Harshbarger | Martinez | Price | Schnoor | DNS | FHSAA 4A Region 1 |
| 3 | Dr. Phillips |
A | Castell | Heredia | Smith | Martin | 4:06.47 | FHSAA 4A District 9 |
| 4 | Wekiva |
A | Patterson | Foster | Medley | Holmes | 3:57.85 | FHSAA State Track & Field Championships (4A / Para-Athlete) |
| 5 | East Ridge |
A | Terrio | Mustin | Terrio | Benson | 4:03.10 | FHSAA 4A Region 1 |
| 6 | Boone |
A | Steinberg | Santana | Robbins | Rowe | 4:08.98 | 4:08.98 Metro East Championship |
| 7 | Master's Academy (Oviedo) |
A | Brown | Silver | Gogley | Slater | 4:20.00 | |
| 8 | Edgewater |
A | Notaro | Hutchinson | Barkett | Spann | 4:31.65 | Poinciana Relay Meet |