| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Sumner High School | A | Casimere | Cooke | Hill | Ford | 1:04.00 | Verified |
| 2 | Metro High School | A | Dixon | Grimes | Jones | Griffith | 55.19 | Verified |
| 3 | Lift for Life Academy Charter Hi | A | Williams | Lindsey | Taylor | Fisher | 53.75 | Verified |
| 4 | Gateway High School | A | Brown | Cannamore | Gray | Nevils | 54.11 | Verified |
| 5 | McKinley Classical Leadership Hi | A | Galarza | Jones | Kamara | Kamara | 59.11 | Verified |
| 6 | Collegiate School of Medicine an | A | Taylor | Marshall | Vaughn | VandenBrink | 59.37 | Verified |
