| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Lake Placid |
A | Felton | Bullard, Jr. | Walton | Williams | DNS | Sebring Relay Meet 2025 |
| 3 | Hardee |
A | Cherry | Myrick | Brunson | Watson | 45.21 | Larry Richardson Memorial Invitational |
| 4 | Okeechobee |
A | Evans | Williams | Stewart | Brifil | 45.29 | Sebring Relay Meet 2025 |
| 5 | Vanguard (LW) |
A | Seymour | Lightborne | Khan | Lacossade | NT |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | LaBelle |
A | Rivas | Hernandez | Davis | Bradshaw | 45.20 | 65th Annual Edison Relays |
| 2 | DeSoto County |
A | Brown | Tice | Wilkinson | Powers | 44.84 | Sebring Relay Meet 2025 |
| 3 | Sebring |
A | Shannon | Pearson | Mitchell | Olds | 43.49 | Avon Park Meet #1 2025 |
| 4 | Clewiston |
A | Mason | Moody | Pope | Harshaw | 44.30 | Kings Academy Varsity 2 |
| 5 | Moore Haven |
A | Gunsby | Ammons | Davis | Adderley | 44.84 | Immokalee Last Chance |