The 18-year-old has been the most prolific batter for five-time champions Chennai, who dropped to seventh place in the 10-team league after Saturday's defeat by Sunrisers Hyderabad.
The top-order batter milked 30 off 12 balls but was seen clutching his hamstring and required lengthy on-field treatment before being dismissed.
"Obviously a hamstring tear. I don't know how bad it is," Chennai batting coach Mike Hussey told reporters after the match.
"I'm sure we'll scan him probably tomorrow or the next day.
"It looks pretty bad, unfortunately. And he's going to be a big, big loss because he's been in really nice touch for us."
Mhatre has scored 201 runs across six innings at a healthy 177-plus strike rate.
"He's an exciting young talent, but it's going to give someone else an opportunity. So that's exciting as well," Hussey said.
"We've got some really good players that haven't been able to get an opportunity. And so it's going to be exciting for one of those guys.
"So, as much as we're really disappointed to lose Ayush - I don't know how long for - it's exciting for a new player to come in and get a chance."
Chennai face Mumbai Indians in their next match on Thursday.