| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Lake Weir |
A | Fisher | HOLIDAY | AGOSTO | 3:43.94 | FHSAA 3A District 5 | |
| 3 | South Lake |
B | Alexis | Stewart | Parker | Clinton | NT | |
| 4 | South Lake |
A | McKee | Swaby | Richards | Smith | 3:22.78 | FHSAA 4A Region 1 |
| 5 | Wildwood |
A | Slowley | Spurrier | Thompson | Corbin | 3:31.72 | FHSAA State Track & Field Championships (1A) |
| 6 | Lake Weir |
B | CORREIA | SMITH | WILLIS | Henderson | NT | FHSAA 3A District 5 |
| 7 | Vanguard (Ocala) |
A | NT |