| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Winter Springs |
A | 43.43 | Bishop Moore - Larry Kelly Invitational 2025 |
| 2 | Lake Brantley |
A | 43.11 | Charlie Harris Relay |
| 3 | Lake Mary |
A | 42.53 | Lake Mary Invitational 2025 |
| 4 | Lake Howell |
A | 42.13 | Charlie Harris Relay |
| 5 | Oviedo |
A | 42.52 | Lake Mary Invitational 2025 |
| 6 | Hagerty |
A | 42.70 | ROYAL INVITATIONAL @ The First Academy 2025 |
| 7 | Seminole (Sanford) |
A | 43.34 | George Janan Spruce Creek Invitational |
| 8 | Crooms |
A | 51.04 | Coach Ernest McPherson Invitational 2025 |