| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed |
|---|---|---|---|---|---|---|---|
| 2 | Master's Academy (Oviedo) | A | Wilson, Ryleigh | Wilson, Alexa | Falco, Ana | Jacobus, Lilly | NT |
| 3 | Winter Park | A | Schultz, Anamaria | White, Mallory | Francis, Abagail | Gonzalez, Sophia | 53.50 |
| 4 | Lake Mary | A | Petty, Knielle | Juarez, Karina | Upshaw, JaNiya | Gaspar, Nellysa | 52.39 |
| 5 | Dr. Phillips | A | Mendez, Irmiris | Jones, Jayla | Formey, Zanea | Scott, Raina | 49.39 |
| 6 | Timber Creek | A | Reed, Lia | Bolden, Adrianna | Chinn, Zora | Perez, Nadia | 49.94 |
| 7 | Oviedo | A | Wilks, Felicia | Joseph, Juliana | Lofton, Skye | Chandler, Ayden | DQ |
| 8 | Lake Mary Prep | A | Perdue, Kristianne | Barnes, Leilani | Gaston, Nicole | Marshall, Jazmin | DNS |
| 9 | Colonial | A | Bailey, Arianna | Bailey, Zaria | Prek, Paula | Hormazabal, Karla | NT |