| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 4 | Innovation_Orlando | A | DNS | Harmony Invitational |
| 5 | Boone |
A | 3:38.46 | West Orange - Dr. Baptiste Invitational 2025 |
| 6 | Cypress Creek |
A | 3:41.45 | Bears Invitational (Regional Qualifier) |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Freedom (Orlando) |
A | 3:34.53 | Tiger Invitational |
| 3 | East River |
A | 3:29.92 | Embry-Riddle Running Elements Classic High School |
| 4 | University (Orlando) |
A | 3:27.52 | ROYAL INVITATIONAL @ The First Academy 2025 |
| 5 | Timber Creek |
A | 3:22.36 | 2025 The Ellis Elite 16 Track and Field Invitational |
| 6 | Winter Park |
A | 3:22.88 | Charlie Harris Relay |
| 7 | Oak Ridge |
A | 3:27.90 | Oak Ridge - Pioneer Invitational 2025 |
| 8 | Jones |
A | 3:32.70 | Bears Invitational (Regional Qualifier) |
| 9 | Lake Nona |
A | 3:36.27 | Timber Creek National Showcase 2025 |