| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Frostproof |
A | 5:07.07 | Lake Region HS Distance Carnival 2025 |
| 3 | All Saints' |
A | 4:45.39 | Harmony Invitational |
| 4 | Davenport |
A | DNS | Bob English Invitational 2025 |
| 5 | Lakeland |
A | 4:38.45 | Tiger Invitational |
| 6 | Tenoroc |
A | 4:58.93 | Blue Devil Invitational (Regio |
| 7 | Kathleen |
A | DNS |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Bartow |
A | 4:37.22 | ROYAL INVITATIONAL @ The First Academy 2025 |
| 2 | Auburndale |
A | 4:29.79 | Lake Region Sprint Hurdle Night 2025 |
| 3 | Victory Christian (LL) |
A | 4:18.12 | Calvary Christian Warrior Invitational |
| 4 | George Jenkins |
A | 4:01.36 | Berkeley Track and Field Invitational |
| 5 | Winter Haven |
A | 4:17.45 | Blue Devil Invitational (Regio |
| 6 | Discovery |
A | 4:23.31 | 2025 Titan Invitational |
| 7 | McKeel |
A | 4:37.19 | Lakeland City Championships @ George Jenkins 2025 |
| 8 | Lake Region |
A | DNS | Lake Region HS Distance Carnival 2025 |