| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 3 | Mount Dora | A | NT | |
| 4 | George Steinbrenner | A | 45.50 | |
| 5 | Mulberry | A | 46.50 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Haines City | A | 44.60 | |
| 2 | Celebration | A | DNS | |
| 3 | Winter Haven | A | DNF | 2019 FHSAA State Track & Field Championships |
| 4 | Lakeland | A | 41.84 | 2019 FHSAA State Track & Field Championships |
| 5 | Dr. Phillips | A | 42.03 | 2019 FHSAA State Track & Field Championships |
| 6 | Plant City | A | 43.40 | |
| 7 | George Jenkins | A | 44.00 | |
| 8 | Lakeland Christian | A | DNS | St. Cloud Track Invitational |