| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Pasco |
A | Daley | Hill | Ellis | Christensen | NT | |
| 2 | Gulf |
A | Cardoso | Green | Gordon | Quarg | 4:52.12 | FHSAA 3A Region 2 |
| 3 | Wiregrass Ranch |
A | Brown | Knowles | Angulo | Durham | 4:28.20 | 2025 Newsome Wolves Invitational |
| 4 | Tarpon Springs |
A | Jarrell | Sponsel | Raeffer | Shan | 4:19.59 | FHSAA 2A District 9 |
| 5 | South Lake |
A | Manning | Miller | Guzman | Williams | 4:24.47 | Be Like Ike Preseason Opener hosted by Lake Minneola High Sc |
| 6 | Mitchell |
A | Hunter | Hunter | Unger | Little | 4:30.41 | West SAC Conference Meet |
| 7 | Anclote |
A | Wolfinger | Kervin | Wolfinger | Mack | NT |