| 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 |