| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Classical Prep |
A | Brown | Kehoe | Keller | Miranda | NT | River Ridge High School Last Chance Meet |
| 4 | Cypress Creek (Wesley Chapel) |
A | Frazier | Clifton | Knowles | Jackson | 4:00.00 | |
| 5 | Fivay |
A | Rodriguez | Green | Luna | Oliver | 4:11.94 | Anclote Sharks Invitational |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Bayshore |
A | Hughes | Johnson | Alexandre | Youmans | 3:55.70 | 3:55.70 Braden River Invitational |
| 3 | Gulf |
A | Brown | Coleman | Porter-Jackson | Carpenter | NT | 3:35.74 Archer Invitational |
| 4 | Land O'Lakes |
A | Faria | Wells | Vazquez | Lorenzo | NT | FHSAA 4A Region 3 |
| 5 | Wesley Chapel |
A | Lewis | Williams | Adelson | Fuentes | 3:29.95 | FHSAA 3A Region 2 |
| 6 | River Ridge |
A | McCarthy | Smyth | Favero | Williams | 3:41.00 | |
| 7 | Angeline Academy | A | Senevirathne | Petrovich | Lowe | Gross | 3:56.28 | FHSAA 2A Region 3 |