| 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 |