| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Geneva School |
A | Lindsay | Newhartz | Flores | Duvra | NT | |
| 4 | University (OC) |
A | Bryant | Smith | Dykstra | Heard | 55.35 | Sanford-Seminole Open 2025 |
| 5 | Spruce Creek |
A | Sanchez | Stoutamire | McCaskill | Stabile | 56.09 |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Trinity Prep |
A | Burgos | Moore | Yared | Hill | 52.58 | FHSAA 2A Region 2 |
| 2 | Hagerty |
A | Mantilla | Leger | Jones | Ali | 52.00 | |
| 3 | Lake Mary |
A | Carter | Holley | Haynes | Mcclelland | 49.58 | 49.58 Hagerty Track and Field Invitational 2024 |
| 4 | Lake Brantley |
A | Ary | Edwards | White | Nwaogwugwu | 48.76 | FHSAA 4A Region 1 |
| 5 | Seminole (Sanford) |
A | Reid | White | Burgos | Voltaire | 49.00 | |
| 6 | Lake Howell |
A | Ramon | Stokes | Bibb | Bibb | 49.80 | FHSAA 4A Region 1 |
| 7 | Oviedo |
A | Harshbarger | Martinez | Schnoor | Hodge | 52.08 | 52.08 Lake Mary Invitational 2024 %28Regional Qualifer%29 |