| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | University School of Johnson Cit | A | 1:01.00 | Unverified: |
| 2 | West Ridge High School | A | 54.92 | Verified |
| 3 | Daniel Boone High School | A | 54.29 | Verified |
| 4 | Science Hill High School | A | 53.01 | Verified |
| 5 | Blacksburg High School | A | 51.50 | Unverified: |
| 6 | Volunteer High School | A | 52.06 | Verified |
| 7 | Tennessee High School | A | 54.20 | Verified |
| 8 | Patrick Henry (G. Spring) | A | 54.75 | Unverified: Falcon Early Bird Spring Meet |
| 9 | Cherokee High School | A | 56.13 | Verified |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 4 | Tennessee High School | B | NT | Verified |
| 5 | Science Hill High School | B | NT | Verified |
| 6 | Abingdon High School | A | NT | Unverified: |
| 7 | Blacksburg High School | B | NT | Unverified: |