| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed |
|---|---|---|---|---|---|---|---|
| 1 | MENENDEZ |
A | Downey | Bryant | Massey | Goodwin | NT |
| 2 | RIDGEVIEW (OP) |
A | Dixon Jr. | Burdetsky-Smith | Watkins | Marshall | 44.85 |
| 3 | CLAY |
A | Thomas | Blaha | Burke | Phenneger | 44.22 |
| 4 | BISHOP SNYDER |
A | Carney | Jones | Mitchell | Navitsky | 43.91 |
| 5 | ORANGE PARK |
A | Bradley | Campbell | Riley | Richardson | 44.05 |
| 6 | BRADFORD |
A | Hall | Tyson | Williams | Lawrence | 44.36 |
| 7 | MIDDLEBURG |
A | Dawson | Roseme | Gilbert | Ferguson | 46.55 |