| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Mason Classical Academy |
A | Alce | Finn | Grajales-Franco | Mummert | 47.86 | Guy Thomas Memorial Classic (Approved Qualifier) |
| 4 | Donahue Academy |
A | Rankin | Mariano | Patterson | Parra | 45.48 | FHSAA 1A Region 3 |
| 5 | Village School |
A | Martinicchio | Seifert | Haag | Felix | 46.08 | FHSAA 1A Region 3 |