| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Lake Buena Vista |
A | Porter | Comer | Carlucci-Harris | Faria | 54.54 | Wolverine Relays |
| 2 | South Lake |
A | Johnson | Sheffield | Harrison | Williams | 51.92 | Archer Invitational |
| 3 | Edgewater |
A | Harris | Barkett | Hutchinson | Brown | 49.98 | FHSAA 3A Region 2 |
| 4 | Jones |
A | Ocasio | Jones-Graham | Sheffield | Brown | 48.00 | FHSAA State Track & Field Championships (3A) |
| 5 | Cypress Creek |
A | Moss | Nix | Lauterwasser | Rodriguez | DNS | Ocoee Invitational |
| 6 | Evans |
A | Torres | Sanon | Davis | Dorn | 50.95 | Blue Devil Invitational (Regio |
| 7 | Freedom (Orlando) |
A | Davis | Matos | Torres | Brown | 53.40 | Kowboy Invitational |