8 fastest times advance to Finals
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed |
|---|---|---|---|---|---|---|---|
| 1 | Ribault |
A | Hagans | Gardner | Cotton | Stokes | 52.66 |
| 2 | Terry Parker |
A | Dawkins | Graham | Russell | Coffey | 51.35 |
| 3 | Riverside |
A | Branch | Patterson | Robinson | Williams | 50.19 |
| 4 | Episcopal |
A | Bow | Bush | McCoy | Watts | 48.81 |
| 5 | Westside |
A | Fanning | Brown | Marshall | Brown | 49.44 |
| 6 | Baldwin |
A | Blackshear | Blackman | Nelson | Trent | 51.13 |
| 7 | Raines |
A | Walden | Alston | Wilson | Acres | 51.85 |
| 8 | Atlantic Coast |
A | Peguese | Martinez | Tebakouna | Gray | 54.66 |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Atlantic Coast |
A | Peguese | Martinez | Tebakouna | Gray | 49.35 | FHSAA 4A District 2 |
| 3 | Ribault |
A | Hagans | Gardner | Cotton | Stokes | 49.06 | 2023 Chandra Cheeseborough Invitational - High School |
| 4 | Terry Parker |
A | Dawkins | Graham | Russell | Coffey | 52.24 | Wolverine Classic |
| 5 | Episcopal |
A | Bow | Bush | McCoy | Watts | 48.45 | FSU Relays (High Schools) |
| 6 | Westside |
A | Fanning | Brown | Marshall | Brown | 49.23 | 2023 Chandra Cheeseborough Invitational - High School |
| 7 | Raines |
A | Walden | Alston | Wilson | Acres | 50.49 | Gateway Conference Track & Field Championship |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Fernandina Beach |
A | Misciagna | Hays | Roberts | Bell | 52.54 | TFRRS 05/05/22 - 1:00 |
| 3 | Ed White |
A | Pryce | Horne | Bussley | Kirby | 51.50 | Ed White Mini Meet 2 |
| 4 | Riverside |
A | Branch | Patterson | Robinson | Williams | 50.68 | FHSAA 3A District 3 |
| 5 | Baldwin |
A | Blackshear | Blackman | Nelson | Trent | 50.91 | Bob Hayes High School Invitational |
| 6 | First Coast |
A | Credle | King | Marseille | McWhite | DNS | FHSAA 4A Region 1 |
| 7 | Eagles View |
A | Blackwood | Smith | Christie | Bailey | NT |