| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Lake Brantley |
A | 3:35.35 | Charlie Harris Relays |
| 2 | Lake Mary |
A | 3:29.44 | Lake Mary Invitational 2024 (Regional Qualifer) |
| 3 | Mainland |
A | 3:27.50 | 20th Annual - East Coast Classic (FHSAA Sanctioned Meet) |
| 4 | Apopka |
A | 3:22.38 | Pepsi Florida Relays |
| 5 | Seminole (Sanford) |
A | 3:22.39 | Charlie Harris Relays |
| 6 | Lake Howell |
A | 3:28.86 | Charlie Harris Relays |
| 7 | Crooms |
A | 3:50.38 | Hagerty Track and Field Invitational 2024 |
| 8 | Winter Springs |
A | 3:36.50 | Charlie Harris Relays |