| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Apopka |
A | NT | |
| 2 | Lake Highland Prep |
A | 53.05 | |
| 3 | Olympia |
A | 51.41 | Windermere Meet of Champions Classic 2024 |
| 4 | Wekiva |
A | 49.87 | Olympia Titans Invitational |
| 5 | Horizon |
A | 51.05 | Windermere Meet of Champions Classic 2024 |
| 6 | Foundation Academy |
A | 52.67 | Windermere Meet of Champions Classic 2024 |
| 7 | Winter Park |
A | 53.50 | |
| 8 | Windermere Prep |
A | DNF |