The winger limped off in the 58th minute of Brighton and Hove Albion's 3-0 win against Wolverhampton Wanderers after suddenly clutching the back of his left thigh while chasing a ball and going down.
Mitoma reportedly left the stadium on crutches which will be a huge worry for Japan head coach Hajime Moriyasu, who is due to name his World Cup squad on Friday.
"We have to wait for the scan, then we can clarify it," said Brighton manager Fabian Hurzeler when asked by reporters about Mitoma's injury after the match.
"Of course, it didn't look great, but I'm a positive person and I stay positive regarding it.
"It looked like a hamstring injury, but let's wait if it's an injury or not."
Japan will begin their World Cup campaign against the Netherlands in Arlington, Texas on June 14.
They will also face Tunisia and Sweden in Group F.
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