| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Boone |
A | Aldeus | Aldeus | Fuehrer | Hodge | 43.24 | Bishop Moore - Carl Friedland 2026 |
| 4 | South Lake |
A | Parker | Holman | Desir | Pring | 42.91 | Olympia Titans Invitational (Regional Qualifier) |
| 5 | Edgewater |
A | Denmark | Nunn | Moss | Hendry | 43.13 | Bishop Moore - Carl Friedland 2026 |
| 6 | West Oaks |
A | Fulmore | Joseph | Staniclasse | Johnson | DNS | Larry Kelly Invitational 2026 |
| 7 | Ocoee |
A | NT |