| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Winter Haven Christian School | A | Manus | Thompson | Daquino | Philips | 59.06 | Blue Devil Invitational (Regio |
| 4 | McLaughlin | A | Menzie | Robinson | Owens | McGriff | 53.65 | 2025 Polk County Freshman Sophomore Championships |
| 5 | Tenoroc |
A | Prokop | Durr | Fernandez | Green | 55.17 | Blue Devil Invitational (Regio |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Frostproof |
A | McNeil | Jones | Butler | Smith | 53.00 | |
| 3 | Haines City |
A | Boncceur | Jasper | Farquharson | Holmes | 52.08 | Kowboy Invitational |
| 4 | Davenport |
A | Moret Flores | Monestine | Jones | O'Hara | 50.88 | Kowboy Invitational |
| 5 | Auburndale |
A | Everett | Boone | Allen-Chona | Forsett | 51.85 | Lake Region Sprint Hurdle Night 2025 |
| 6 | Sebring |
A | Adkins-Williams | Marine | Madison | Madison | 52.64 | Blue Devil Invitational (Regio |
| 7 | Hardee |
A | Pierre | Jackson | Holley | Nunez | 53.58 | Larry Richardson Memorial Invitational |