| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Breese (Mater Dei) | B | Palma | Palma | Teets | Hilmes | 50.00 | |
| 3 | Edwardsville (Metro-East Lutheran) | A | Daly | Chernianskii | Holmes | Kohlmiller | 45.74 | |
| 4 | Freeburg | A | Murphy | Banks | Frech | Thompson | 44.86 | |
| 5 | Belleville (Althoff Catholic) | A | Nave | Lewis | Done | Coldon | 45.06 | |
| 6 | Breese (Mater Dei) | A | Jourdan | Rakers | Grapperhaus | Murphy | 46.10 |