| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Harmony |
A | 51.44 | 51.44 Orange Belt Conference Championships |
| 2 | St. Cloud |
A | 50.77 | 50.77 Harmony Invitational |
| 3 | Osceola (Kissimmee) |
A | 50.50 | 50.50 Orange Belt Conference Championships |
| 4 | Vero Beach |
A | 48.23 | 48.23 Royal Palm Beach Invitational 2 |
| 5 | Treasure Coast | A | 50.28 | 50.28 28th Annual Warrior Invitational 2024 |
| 6 | Martin County |
A | 50.66 | 50.66 Falcon Classic 2024 |
| 7 | Viera |
A | 51.24 | 51.24 2024 Merritt Island Relays |
| 8 | Centennial |
A | 52.46 | 52.46 SLC Championships 2024 |