| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Trinity Prep |
A | Davis-Rocha | Osleger | Moore | Davidescu | 49.25 | 49.25 Trinity Prep - Saints Invitational 2024 |
| 3 | University (OC) |
A | Hunter | Andrews | Elder | Hayes | 45.78 | 45.78 Deland Wednesday Meet 2024 |
| 4 | DeLand |
A | Apakama | Cook | Wright | Caraballo | DNS | 44.15 Sanford - Seminole COACH ERNEST MCPHERSON INVITATIONAL 2024 |
| 5 | Geneva School |
A | Alilin | Davis | Lauretta | Mitchell | 44.38 | FHSAA 1A Region 2 |
| 6 | Crooms |
A | CAINE | PARKES | PINKNEY | ADICHIRAYIL | DNS | 48.15 Hagerty Track and Field Invitational 2024 |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Seminole (Sanford) |
A | Chaplin | Croom II | McKinney | Beckford | 43.50 | |
| 2 | Lyman |
A | Beason | Thelemague | McGowan | Jefferson | 43.30 | |
| 3 | Lake Brantley |
A | Breedlove | Ayala | Richards | Siddiqui | DNS | FHSAA 4A Region 1 |
| 4 | Oviedo |
A | McNeil | Galarza | Slivinski | Nixon | 42.53 | 42.53 Charlie Harris Relays |
| 5 | Lake Mary |
A | Rosario | Dixon | Williams | Soto | 42.68 | FHSAA 4A Region 1 |
| 6 | Lake Howell |
A | Spells | Funnye' | Patterson | Wilkerson | 43.24 | 43.24 SAC Championships 2024 |
| 7 | Spruce Creek |
A | Oxendine | Edwards | Lucas | Williams | DNS | 43.42 Satellite HS Scorpion Invitational 2023 |
| 8 | Hagerty |
A | Davis | Hatfield | Gandy | Howell | 43.77 | Sanford-Seminole Open 2025 |