| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Academy Lakes | A | NT | |
| 2 | Seffner Christian | A | 53.98 | Calvary Christian Warrior Invite 2021 |
| 3 | Berkeley Prep | A | 51.28 | Mustang Invitational |
| 4 | Calvary Christian (Clr) | A | 50.15 | Berkeley Track and Field Invitational |
| 5 | Admiral Farragut | A | 50.16 | Calvary Christian Warrior Invite 2021 |
| 6 | Victory Christian (LL) | A | 52.04 | Lakeland City Meet 2021 |
| 7 | Northside Christian | A | 56.05 | Mustang Invitational |
| 8 | Pepin Academy | A | NT |