| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Glades Central |
A | Frazier | Patrick | Fowler | Sellers | 4:56.68 | PBGHS Invitational 2025 |
| 3 | John I. Leonard |
A | Augustin | lapointe | graham | dorceus | 4:27.63 | John I Leonard Invitational |
| 4 | Pahokee |
A | Johnson | Johnson | White | Richardson | 4:03.59 | Jeff Fossen Invitational 2025 |
| 5 | Royal Palm Beach |
A | Neptune | sainvil | Bradwell | Ennis | DNS | PBGHS Invitational 2025 |
| 6 | Clewiston |
A | Johnson | Scuderi | Miscaide | Bradford | DNS | 65th Annual Edison Relays |
| 7 | Glades Day |
A | Haryman | Carotenuto | LongChamps | Wallace | 5:41.48 | Pahokee Anquan Boldin Invitational 2025 |