| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 4 | Freedom (Orlando) |
A | 4:33.19 | Kowboy Invitational |
| 5 | Lake Nona |
A | 4:28.42 | Timber Creek National Showcase 2025 |
| 6 | Innovation_Orlando | A | 4:29.64 | Lake Nona - Tammy Lafayette Relays 2025 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Boone |
A | 4:21.94 | Viera High School Invitational |
| 3 | Oak Ridge |
A | 4:19.30 | Oak Ridge - Pioneer Invitational 2025 |
| 4 | Jones |
A | 4:18.01 | Tiger Invitational |
| 5 | Timber Creek |
A | 3:59.23 | 2025 The Ellis Elite 16 Track and Field Invitational |
| 6 | Cypress Creek |
A | 4:07.05 | St. Cloud Invitational |
| 7 | Winter Park |
A | 4:18.04 | Timber Creek National Showcase 2025 |
| 8 | East River |
A | 4:20.88 | Guy Thomas Memorial -Sponsored by Anderson Electric of SWFL |