Kemp had an MRI on Thursday. He said Wednesday this injury felt worse than the original one and expects to be out a month, which would mean sometime just before the All-Star break.
The club promoted Alex Castellanos (who returned to Triple-A Albuquerque last week from a hamstring strain) to replace Kemp.
"It seems like everything we were doing was working, and lately nothing seems to be working," said Mattingly, whose team compiled the best record in baseball but now has its first three-game losing streak along with a wave of injuries.
"We felt a letdown tonight when Matt goes down, we got flat. We have to buckle up and get ready to play. Nobody will feel sorry for us. They'll look at us as a team that's banged up and we'll look at us as a team that's going to survive."
Kemp said he felt the hamstring "jab" as he approached third base while scoring from first on a first-inning double by Andre Ethier.
"It's hurting pretty good right now," Kemp said. "It feels worse than the first time. I hope the MRI comes back good and we can go from there. This happens in sports. It happens when you're fast."
Losing pitcher Clayton Kershaw summed up the mood in the clubhouse.
"It's frustrating for everybody," Kershaw said. "We all feel bad for Matt. I know nobody feels worse than him. He wants to play bad. It's just tough. It's never easy for the team to deal with when your superstar is out. To see him go through that is never easy, but at the same time, games keep coming and we've just got to keep grinding and keep playing. This is our first tough stretch of the year. We'll come out of it and be better off for it."
Kemp originally was injured in Chicago on May 4, feeling his leg "pull a little bit" cutting off a ball in the left-center gap.
He was a late scratch May 6, started the next six games playing safety first until finally leaving in the third inning May 13. He went on the disabled list May 14, snapping the longest active streak for consecutive games played at 399.
He played two games in a rehab assignment with Triple-A Albuquerque on Sunday and Monday and was activated for Tuesday night's game, missing the minimum 15 days. The Dodgers went 9-5 while he was disabled.
Kemp disputed the suggestion that he came back too soon.
"No, we went through all the tests, I sprinted real hard, everything was normal," he said. "You don't know what can happen. It just gave out on me. Today it was something different. Something was still in there. It's definitely frustrating. I was so happy being back, now I have to sit awhile."
Mattingly didn't blame Kemp.
"Matt told us he didn't feel anything and I believe him," Mattingly said. "He was way too upbeat and positive. It looked like he was looking for another gear when the ball got away from the center fielder and when he came around third base he didn't look right to me. Hamstrings are tough to figure out. [These injuries are] hard to get rid of and hard to tell when they're gone."
In his first game back Tuesday night, Kemp went 1-for-4 with a double and a nubber in front of the plate that required him to sprint to first with no ill effects. Wednesday night he walked with two outs in the first inning and Ethier followed with a double to the fence in left-center.
Kemp rounded second with the intent to score and was waved home by third-base coach Tim Wallach, but as soon as Kemp hit third base he gradually pulled himself up and scored standing as the throw went to third base.
Kemp's body language as he reached the dugout indicated not all was well. He walked the length of the dugout, followed by two trainers, then grabbed a bat and snapped it over his knee and headed for the clubhouse.
"I didn't know what happened at first, but I figured he got hurt again," Milwaukee's Corey Hart said. "He went nuts."
"I tried to hide it but there was no hiding that from Donnie B," Kemp said. "He asked if I felt something and then he said, 'You're done.' It's not his fault I hurt my leg. I'm not mad at Donnie B. He's protecting his player. He can't throw me out there hurt."
Prior to the injury, Kemp was leading the National League in just about every offensive category, seemingly en route to the MVP award he didn't receive last year. Now he faces being shut down just to salvage the second half in one piece.
"This is the first time I've ever had a hamstring problem and I know they're tricky, they can come back," he said. "I have to take it slow and let it heal. It felt good, it really did. I thought I was 100 percent, but it's still lingering."
Ken Gurnick is a reporter for MLB.com. This story was not subject to the approval of Major League Baseball or its clubs.