| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Lincoln Park |
A | 46.81 | SLC Championships 2025 |
| 2 | Martin County |
A | DNF | Cobra Relays 2025 |
| 3 | Tradition Prep | A | 43.87 | Bulldog Invitational 2025 |
| 4 | Jupiter |
A | 43.62 | Benjamin Invite #2 2025 (Sanctioned) |
| 5 | Fort Pierce Central |
A | 43.75 | Cobra Relays 2025 |
| 6 | Fort Pierce Westwood |
A | DNS | Cobra Relays 2025 |
| 7 | South Fork |
A | 46.20 | Bulldog Invitational 2025 |