| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | St. Edward's |
A | Perrin | Callaghan | Hebig | Elliott | 4:42.40 | Benjamin Invite #2 2025 (Sanctioned) |
| 3 | Fort Pierce Central |
A | Simeus | Fortin | Andrews | Johnson-Clark | 4:23.48 | Cobra Relays 2025 |
| 4 | Centennial |
A | Mitton | Schmook | Jean | Lindo | 4:09.25 | Dr. Garcia Invite #2 2025 |
| 5 | Martin County |
A | Cantu | Lindsay | Snyder | Rapaport | 4:21.23 | Cobra Relays 2025 |
| 6 | Lincoln Park |
A | Whilby | Daniel | Brown | Anderson | 4:32.60 | Cobra Relays 2025 |