| Lane | # | Athlete | Yr. | Team | Seed |
|---|---|---|---|---|---|
| 1 | 239 | JACK, Rakeel | SR | North Florida Speed Track Club | 49.50 |
| 2 | 286 | ROBINSON, Christopher | SR | Rickards HS | 49.17 |
| 3 | 164 | CHUPARKOFF, Christopher | JR | IMG Elite | 48.90 |
| 4 | 271 | WILLIAMS, Treyvon | SR | Palatka HS | 47.56 |
| 5 | 75 | JONES, Jessiah | SO | Empire Athletics | 48.80 |
| 6 | 252 | KAY, Alex | SR | Ocala Vanguard HS | 49.10 |
| 7 | 565 | HEREMAN, Kevin | JR | Wind Speed Athletics | 49.24 |
| 8 | 230 | GAMMONS, Mekhi | 8 | Miami Gardens Xpress Track Club | 49.63 |