| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Boone |
A | Chambers | Drummond | Haynes | Rivers | 3:29.01 | FHSAA 4A District 5 |
| 3 | South Lake |
A | McKee | Stewart | Swaby | Alexis | 3:22.78 | FHSAA 4A Region 1 |
| 4 | Ocoee |
A | Caldwell | Cross | Shakes | Paul | 3:20.31 | FHSAA 4A Region 1 |
| 5 | Edgewater |
A | Casanas | Harris | Sauveur | McKenzie | 3:21.62 | FHSAA 3A Region 2 |
| 6 | Jones |
A | Docteur | Hall | Coleman | Thompson | 3:27.95 | FHSAA 3A Region 2 |
| 7 | West Oaks |
A | Anderson | Williams | Cole | Ards | 3:58.99 | Evans Development Invitational |