The Hobart Fire Department responded to the crash, which involved a car and a semi, on southbound Interstate 65 overnight.
First responders found three people who died at the scene and two people with critical injuries.
A helicopter took one patient to a regional trauma center, and an ambulance transported the other patient to a local hospital for emergency treatment, fire officials said.
What led up to the crash was not immediately clear.
Authorities are investigating. Police did not immediately provide further information about the crash.