| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Deltona |
B | Ciprian | Williams | Cockfield | Alvarez | NT | |
| 4 | Pine Ridge |
B | Coffy | Fedrick | Colon | Scott | NT | |
| 5 | University (OC) |
B | Rodriguez Velazquez | Rushing | Harris-El | Evans | NT |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | University (OC) |
A | Bass | Garcia | Hough | Williams | 55.35 | Sanford-Seminole Open 2025 |
| 3 | Deltona |
A | Andre | Petit Do | Eckford | Falcone | 51.17 | George Janan Spruce Creek Invitational |
| 4 | Mainland |
A | Watlington | Gillon | Joseph | Lett | 50.78 | |
| 5 | Pine Ridge |
A | Braggs | Thompson | Savius | Gorham | 51.11 | 51.11 WOLVES SPECTACULAR |
| 6 | Seabreeze |
A | Olsen | Ann | De Jesus | Hick | 52.07 | Cocoa Tigers Relays Invitational |
| 7 | Trinity Christian (Deltona) |
A | Rodriguez | Mutty | Easter | Marrow | DNS | Bishop Moore - Carl Friedland Invite 2025 |