| Lane | Athlete | Team | Seed |
|---|---|---|---|
| 2 | CARROLL, Christian | Bartram Trail | 44.50 |
| 3 | LANGELAND, Connor | Bartram Trail | 42.13 |
| 4 | GOMBAR, Caleb | Bartram Trail | 41.06 |
| 5 | MONTERO, Enrico | Fleming Island | 41.72 |
| 6 | HAMMETT, Jaycob | Bartram Trail | 42.80 |
| 7 | SAVAGE, Avery | Fleming Island | NT |