| 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 |