| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | McLaughlin | A | Madera | Menzie | Owens | McGriff | DNS | Frostproof - The Ridge Open 2025 |
| 4 | Davenport |
A | Neal | Blanquicet | Moret Flores | Jones | 4:38.29 | Bob English Invitational 2025 |
| 5 | Tenoroc |
A | Henao | Fernandez | Alfonso | Adams | 4:46.09 | FHSAA 2A Region 3 |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | All Saints' |
A | Yoder | Hawkins | Cook | Kamara | 4:37.90 | FHSAA 1A Region 3 |
| 2 | Bartow |
A | Fernandez | Richards | Bourdon | Shakan | 4:25.66 | 4:25.66 TFA - Royal Invitational 2024 %28Regional Qualifier%29 |
| 3 | Auburndale |
A | Payoute | Forsett | Arciniega | Smith | 4:21.25 | FHSAA 3A Region 2 |
| 4 | Winter Haven |
A | Davis | Weaver | Miller | Leverston | 4:03.17 | Blue Devil Invitational (Regional Qualifier) |
| 5 | Haines City |
A | Morris | Morris | Cote | Soler Serrano | 4:10.00 | |
| 6 | Sebring |
A | Melendez | Madison | Marine | Adkins-Williams | 4:21.61 | FHSAA 3A Region 2 |
| 7 | Hardee |
A | Holley | Taylor | Herrera | Nunez | 4:27.75 | Avon Park Meet #1 2025 |