| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | DeSoto County |
A | DNS | 47.85 Sebring Relays 2023 |
| 2 | Lakewood Ranch |
A | 45.00 | |
| 3 | Southeast |
A | 43.76 | 43.76 Riverview Ram Invite 2023 |
| 4 | Manatee |
A | 41.45 | FHSAA Class 4A Track & Field State Championships |
| 5 | Lennard |
A | 42.82 | 42.82 2023 Manuel-Griffin-Fraundorfer Relays |
| 6 | Palmetto |
A | 43.95 | 43.95 FSU Relays %28High Schools%29 |
| 7 | Braden River |
A | 45.68 | 45.68 59th ANNUAL EAGLE INVITATIONAL |
| 8 | Out-of-Door |
A | DNS |