| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Spr. Shawnee | B | Johnson | Baltimore-Brown | Flannery | Frost | NT | |
| 4 | Spr. Northwestern | A | Leaver | Black | Smith | Chiappetta | 1:02.69 | |
| 5 | Mad. Plains | A | Riffle | Orahood | Barajas | Eldridge | 1:09.08 | |
| 6 | London | B | Martin | Edley | Montes-Rojas | Bracho | NT |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | West Jefferson | A | Bias | Headington | Eitel | Bendure | 1:00.41 | |
| 2 | Kenton Ridge | A | Whatley | Glass | Pennington | Adams | 58.69 | |
| 3 | Bellefontaine | A | Jones | Meregote | Bosiljevac | Kuhns | 58.40 | |
| 4 | WCH Washington | A | Tyree | Anderson | Bobst | Mitchell | 57.26 | |
| 5 | London | A | Miller | Guess | Fields | Lohrer | 57.77 | |
| 6 | WCH Washington | B | Vanderbilt | Coleman | Preston | Camp | 58.40 | |
| 7 | Spr. Shawnee | A | Johnson | Books | Leis | Mcgee | 59.87 | |
| 8 | Stivers School For The Arts | A | Hunter | Strodes | Beasley | Chappel | 1:00.42 |