| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Martin County |
A | Dennis | Kideys | Gilbride | Allison | 44.96 | Cobra Relays 2025 |
| 4 | Centennial |
A | Rodriguez | Michel | Hylton | Johns | 40.96 | Bill Wilson Invitational 2025 |
| 5 | Fort Pierce Central |
A | Tommie-Frierson | Hills | Watts | Flowers | 43.75 | Cobra Relays 2025 |
| 6 | St. Edward's |
A | Niemi | Donohoe | Dambournet | Cocks | 49.46 | Benjamin Invite #1 2025 (Sanctioned) |