| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | St. John Neumann |
A | DNS | |
| 3 | South Fort Myers |
A | 4:38.79 | 4:38.79 2023 South Fort Myers Wolfpack Invitational |
| 4 | Cape Coral |
A | 4:40.00 | |
| 5 | Bonita Springs |
A | 5:00.26 | 7th Annual Loretta Purish Invitational-Ladies Only |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Aubrey Rogers | A | 4:28.93 | 60th Annual Eagle Invitational |
| 2 | Palmetto Ridge |
A | DNS | 4:26.32 2023 South Fort Myers Wolfpack Invitational |
| 3 | Southwest Florida Christian |
A | 4:24.00 | |
| 4 | Oasis |
A | 4:24.84 | FHSAA 2A Region 3 |
| 5 | Ida Baker |
A | 4:27.89 | FHSAA 3A District 11 |
| 6 | LaBelle |
A | 4:38.57 | 4:38.57 Sebring Relays 2023 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | North Fort Myers |
A | 4:14.67 | FHSAA 3A Region 3 |
| 2 | Bishop Verot |
A | DNS | FHSAA 2A Region 3 |
| 3 | Barron Collier |
A | 4:01.81 | FHSAA Class 3A Track & Field State Championships |
| 4 | Fort Myers |
A | 4:09.00 | |
| 5 | Naples |
A | 4:12.21 | |
| 6 | Canterbury (FM) |
A | 4:15.98 | FHSAA 1A District 12 |