| Lane | Team | Squad | Seed | Note | 
|---|---|---|---|---|
| 2 |  Bishop Verot | 
		A | NT | |
| 3 |  Ida Baker | 
		A | NT | |
| 4 |  Evangelical Christian | 
		A | NT | 
| Lane | Team | Squad | Seed | Note | 
|---|---|---|---|---|
| 1 |  Estero | 
		A | 1:00.00 | |
| 2 |  Mason Classical Academy | 
		A | 56.96 | |
| 3 |  LaBelle | 
		A | 55.39 | 55.39 Sebring Relays 2023 | 
| 4 |  St. John Neumann | 
		A | 56.00 | |
| 5 |  Gateway Charter | 
		A | 57.91 | FHSAA 2A District 12 | 
| Lane | Team | Squad | Seed | Note | 
|---|---|---|---|---|
| 1 |  Barron Collier | 
		A | 54.93 | CSN Invitational Powered by UnderArmour | 
| 2 |  Oasis | 
		A | 53.09 | FHSAA 2A Region 3 | 
| 3 | Aubrey Rogers | A | 52.78 | 60th Annual Eagle Invitational | 
| 4 |  South Fort Myers | 
		A | 52.97 | 52.97 2023 South Fort Myers Wolfpack Invitational | 
| 5 |  Fort Myers | 
		A | 54.30 | |
| 6 |  Bonita Springs | 
		A | 55.11 | FHSAA 1A District 8 | 
| Lane | Team | Squad | Seed | Note | 
|---|---|---|---|---|
| 1 |  Cape Coral | 
		A | 52.43 | 52.43 Lee County Championships | 
| 2 |  Canterbury (FM) | 
		A | 50.37 | FHSAA Class 1A Track & Field State Championships | 
| 3 |  North Fort Myers | 
		A | 49.11 | 49.11 2023 South Fort Myers Wolfpack Invitational | 
| 4 |  Naples | 
		A | 50.24 | |
| 5 |  Palmetto Ridge | 
		A | 51.08 | FHSAA 4A District 8 | 
| 6 |  Southwest Florida Christian | 
		A | 52.45 |