| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 3 | Pine Ridge | A | DNS | |
| 5 | New Smyrna Beach | A | DNS |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | University (OC) | A | 3:43.23 | 3:43.23 Coach Ernest McPherson Invitational @ Sanford Seminole High |
| 2 | Horizon | A | 3:40.00 | |
| 3 | Mainland | A | 3:30.00 | |
| 4 | Lake Minneola | A | 3:22.01 | 3:22.01 Windermere Meet of Champions Classic 2022 |
| 5 | Eustis | A | 3:28.51 | FHSAA 2A Region 2 |
| 6 | North Marion | A | 3:39.40 | Early Hawk Invitational |
| 7 | Lake Weir | A | 3:42.87 | Trinity Catholic Tuesday Invitational |
| 8 | Father Lopez | A | 3:44.18 | East Coast Classic |