| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 3 | George Steinbrenner |
A | DNS | Steinbrenner Quad Meet |
| 4 | Alonso |
A | 52.26 | Kincaid Classic 2024 |
| 5 | Mitchell |
A | 53.63 | Coyote Invite Cypress Creek Wesley Chapel |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Haines City |
A | 51.89 | Berkeley Track and Field Invitational |
| 2 | Bloomingdale |
A | 51.70 | Nash Higgins Invitational 2024 |
| 3 | Tarpon Springs |
A | 50.13 | FHSAA 2A Region 3 |
| 4 | Hillsborough |
A | 49.43 | George Jenkins - Bob English Invitational 2024 |
| 5 | Wharton |
A | 50.11 | Newsome Wolves Invitational 2024 |
| 6 | Wiregrass Ranch |
A | 50.55 | TFRRS 02/10/23 - we are going to run 53.0 at the fastest |
| 7 | Berkeley Prep |
A | 51.76 | Calvary Christian Warrior Invitational QUALIFIER |
| 8 | Springstead |
A | 52.11 | Weeki Wachee Invitational 2024 |