| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 3 | Harmony |
A | 46.97 | 46.97 Mike Clemens Classic %28Kowboy%29 |
| 4 | Melbourne |
A | 43.78 | 43.78 Kowboy Invitational |
| 5 | St. Cloud |
A | DNS | 44.06 Kowboy Invitational |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Fort Pierce Central |
A | 43.63 | 43.63 Cobra Relays 2024 |
| 3 | Vero Beach |
A | 42.76 | 42.76 Jensen Beach / Chandler Wealth Invitational 2024 |
| 4 | Viera |
A | 41.91 | 41.91 Cape Coast Conference Championship 2024 |
| 5 | Osceola (Kissimmee) |
A | 42.70 | 42.70 Kowboy Invitational |
| 6 | Treasure Coast | A | 43.45 | 43.45 South Fork Meet %232 2024 |
| 7 | Centennial |
A | 43.67 | 43.67 SLC Championships 2024 |