| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Crooms |
A | NT | |
| 2 | Lake Brantley |
A | 50.85 | Olympia Titans Invitational |
| 3 | Lake Howell |
A | 50.55 | 2024 George Janan Spruce Creek Invitational (FHSAA SACTIONED |
| 4 | Seminole (Sanford) |
A | 48.66 | Sanford - Seminole Invitational 2024 |
| 5 | Hagerty |
A | 49.26 | Bishop Moore - Larry Kelly Invite 2024 (Regional Qualifier) |
| 6 | Lake Mary |
A | 50.57 | Harmony Invitational |
| 7 | Winter Springs |
A | 51.79 | Charlie Harris Relays |