| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Calvary Christian (PtStL) | A | Morales | Clark | Gonzalez | Brown | 59.16 | Bulldog Invitational 2025 |
| 3 | Okeechobee |
A | Aponte | Hebel-Hoover | Nesby | Rochelle | 55.08 | Satellite High Reggie Johnson Invite |
| 4 | Jensen Beach |
A | Peruski | Brown | Laurent | Feurer | 54.89 | Benjamin Invite #1 2025 (Sanctioned) |
| 5 | Jensen Beach |
B | Carter | Bradley | Bhatt | Garbellano | 54.89 | Benjamin Invite #1 2025 (Sanctioned) |
| 6 | St. Edward's |
A | Gayle | Perez | Jensen | Elliott | 55.43 | Viera High School Invitational |
| 7 | Treasure Coast Classical | A | Brooks | Burns | Kellerman | Babi | 1:02.05 | MCHS Fesh/Soph 2025 |