| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Winter Haven |
A | Booker | Handofrd | Alls | Kearney | DQ | FHSAA 4A Region 2 |
| 2 | Bartow |
A | White | Robinson | Robinson | Medaries | 3:36.74 | Freedom Spring Break Invitational 2025-Sanctioned |
| 3 | Hardee |
A | Parker | Brunson | Thompson | Myrick | 3:35.42 | FHSAA 2A Region 3 |
| 4 | Tenoroc |
A | Adams | Douglas | Furman | Thomas | 3:33.45 | Lakeland City Championships @ George Jenkins 2025 |
| 5 | Frostproof |
A | Miller | Ramirez | Robey | Shields | 3:51.84 | Frostproof - The Ridge Open 2025 |
| 6 | Sebring |
A | Mendoza | Barefield | Hamilton | Volvaire | 3:36.27 | Sebring Relay Meet 2025 |
| 7 | Haines City |
A | Andrews | Rodriguez | Brooks | Davy | 3:40.51 | Bob Hayes High School Invitational |
| 8 | Davenport |
A | Dabrio | Snow | Key | Medina Palencia | 3:50.13 | Kowboy Invitational |