| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 1 | Fredericktown | B | Streby | Miller | Overholt | Randall | NT | |
| 2 | Centerburg | B | Parker | Kish | Sands | Hernandez-Beltran | NT | |
| 3 | Centerburg | A | Davis | Ballinger | Miles | Quick | 56.22 | |
| 4 | Fredericktown | A | Horlacher | Thompson | Reiss | Meszarosova | 55.52 | |
| 5 | Danville | A | Samples | Brecheisen | McGrady | Staats | 55.64 | |
| 6 | Wester. Genoa Christian | A | Hammond | Herbruck | Kinley | Mangerchine | 55.84 | |
| 7 | Centerburg | C | Wilhelm | Tabor | Waddell | Tabor | DQ |