| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | Santa Fe Catholic |
A | DNS | 2025 Titan Invitational |
| 3 | Tenoroc |
A | 3:40.28 | Bob English Invitational 2025 |
| 4 | George Jenkins |
A | 3:32.78 | 2025 Newsome Wolves Invitational |
| 5 | Lakeland |
A | 3:36.15 | Bob English Invitational 2025 |
| 6 | Lake Gibson |
A | 3:53.72 | 2025 Polk County Freshman Sophomore Championships |
| 7 | Kathleen |
A | NT |