| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 3 | Master's Academy (Oviedo) |
A | NT | |
| 4 | Winter Park |
A | 54.18 | Sanford-Seminole Open 2025 |
| 5 | University (OC) |
A | DNF | Sanford-Seminole Open 2025 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Circle Christian |
A | 52.07 | FHSAA 1A Region 2 |
| 2 | Winter Springs |
A | DNS | FHSAA 3A Region 2 |
| 3 | Mainland |
A | 49.66 | FHSAA 3A District 6 |
| 4 | Lake Brantley |
A | 48.76 | FHSAA 4A Region 1 |
| 5 | Seminole (Sanford) |
A | 49.00 | |
| 6 | Lake Howell |
A | 49.80 | FHSAA 4A Region 1 |
| 7 | Father Lopez |
A | 51.68 | FHSAA 1A Region 2 |
| 8 | Oviedo |
A | DNS | 52.08 Lake Mary Invitational 2024 %28Regional Qualifer%29 |