| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Hardee |
A | Nadaskay | Parker | Benitez | Lopez | 8:46.73 | Larry Richardson Memorial Invitational |
| 2 | Sebring |
A | Mercer | Turner | Hicks | DeJesus | 9:00.00 | |
| 3 | Tenoroc |
A | Adams | Manning | Vasquez | Cavinder | 9:22.95 | 2025 Polk County Freshman Sophomore Championships |
| 4 | Davenport |
A | Joiner | Locker | Rodriguez | Medina Palencia | 9:23.01 | 2025 Polk County Freshman Sophomore Championships |
| 5 | Haines City |
A | Joseph | Ramirez | Scruggs | Denton | 9:41.68 | Kowboy Invitational |
| 6 | Frostproof |
A | Rocendo | Sanchez Aguirre | Griffiths | Ramirez | 9:48.27 | Frostproof - The Ridge Open 2025 |