| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Calvary Christian (Clr) | A | 44.63 | Mustang Invitational |
| 3 | Berkeley Prep | A | 44.22 | Warhawk Invitational |
| 4 | Lakeland Christian | A | 43.53 | Berkeley Track and Field Invitational |
| 5 | Northside Christian | A | 43.97 | Indian Rocks Cavonis Invitational |
| 6 | Victory Christian (LL) | A | 44.36 | Lakeland City Meet 2021 |
| 7 | Indian Rocks Christian | A | 44.75 | Mustang Invitational |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 3 | Pepin Academy | A | 48.96 | Indian Rocks Spike Night 2 |
| 4 | Academy Lakes | A | 46.82 | Mustang Invitational |
| 5 | Admiral Farragut | A | 47.84 | Mustang Invitational |