| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Freedom (Orlando) |
A | Rijos | Guevara | Soyez | Thompson | 3:39.45 | Kowboy Invitational |
| 3 | Lake Buena Vista |
A | Zuluaga | Ferace | Pasteur | Cenemat | 3:34.46 | FHSAA 3A Region 2 |
| 4 | Edgewater |
A | Vecchio | Cleveland | Casanas | Collin | 3:28.38 | FHSAA 3A Region 2 |
| 5 | South Lake |
A | Stewart | Richards | Parker | McKee | 3:33.45 | Ocoee Invitational (Meet is Full) |
| 6 | Cypress Creek |
A | Brice | Challenger | Mathews | Saint Fort | 3:35.61 | FHSAA 4A District 9 |
| 7 | Jones |
A | Laster | Jacobs | Johnson | Hall | FS | Cypress Creek Early Season Meet 2025 |