| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Donahue Academy |
A | Iriarte | Stonehouse | Graham | Radonis | 56.96 | CSN Invitational Powered by UnderArmour (Qualifier) |
| 4 | Village School |
A | Bidwell | Lampkin | Lappin | Dooney | 53.06 | FHSAA 1A Region 3 |
| 5 | Mason Classical Academy |
A | Demezier | Mummert | Pierre Louis | Pierre-Louis | 54.32 | Patriots Friday Night Lights |