| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 3 | Mount Vernon Nazarene |
A | Couchman | Skinner | Conner | Near | 44.03 | Reggie Thomas Classic- Tiffin |
| 4 | Indiana Wesleyan |
A | Wilkins | Parker | Morgan | Torok | 43.18 | IWU Polar Bear Invite |
| 5 | Grace |
A | Findley | Green | Williams | Smith | 42.14 | Manchester Invitational |
| 6 | Huntington |
A | Meek | Atkins | Bise | Felger | 42.66 | Warrior Opener |
| 7 | St. Francis (Ind.) |
A | Malone | Gordon | Forker | Plattner | 43.72 | 2021 George Glass Invitational (Taylor University) |
| 8 | Indiana Kokomo |
A | Dell | Kottkamp | Cooper | Turner | DNF | Indiana Little State Championships |