| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | LaBelle | B | Collins | Garcia | Esquivel | Vasquez | 56.21 | Lake Placid Meet 2 |
| 3 | Sebring | B | Brown | Hood | Freeland | Davis | 54.54 | 2023 Hardee Wildcat Relays |
| 4 | LaBelle | A | Sandoval | Chairez | Solis | Wheatle | 56.21 | Lake Placid Meet 2 |
| 5 | Gateway Charter | A | Rayner | Madhere | Thomas | Louis | 57.92 | TITAN MINI-MEET (hand-timed--no hurdles) |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Okeechobee | A | Aponte | Nesby | Hancock | Lamb | 55.10 | South Fork Meet #1 |
| 2 | Hardee | A | Holley | Holley | Mushrush | Pierre | DQ | 2023 Hardee Wildcat Relays |
| 3 | Glades Day | A | Willis | George | Sturgis | Lohmann | 53.74 | Glade Central Invitational 2023 |
| 4 | Sebring | A | Hawthorne | Jones | Davis | Ramberger | 54.54 | 2023 Hardee Wildcat Relays |
| 5 | Avon Park | A | Brassell | Watts | Davis | Atkins | 54.81 | Lake Placid Meet 3 |
| 6 | Immokalee | A | Thelisma | Short | Jean | Green | 55.20 | 9th Annual Loretta Purish Invitational |