| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Victory Christian (LL) |
A | McGrath | Jackson | Palavinici-Gomez | Freeman | 1:01.10 | |
| 4 | Trinity Christian (Deltona) |
A | Toman | Jeanclaude | Easter | Munn | 57.38 | Mount Dora Christian Invitational Meet #2 |
| 5 | Hagerty |
A | Maboudou-Tchao | Basilo | Hanus | Sauvao | 1:00.00 |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Geneva School |
A | Rawlings | Cota | Gonzales | Trifone | 50.55 | 50.55 Mount Dora Christian Invitational Meet %232 |
| 3 | Father Lopez |
A | Fulton | Loach | Benedict | Frances | 51.68 | FHSAA 1A Region 2 |
| 4 | Lake Howell |
A | Adams | Postell | Mazumba | Scott-Bernard | 49.80 | FHSAA 4A Region 1 |
| 5 | First Academy (Orlando) |
A | Harris | Jackson | Mercedes | Schmid | 51.66 | ROYAL INVITATIONAL @ The First Academy 2025 |
| 6 | Cornerstone Charter |
A | Cuevas Serrano | Harman | Mitchell | Roberts | 56.00 | |
| 7 | Lake Mary |
A | Brooks | Mandujano | Allen | Chester | 49.58 | 49.58 Hagerty Track and Field Invitational 2024 |