Dutch winger Arjen Robben will undergo a scan on his hamstring which will determine his World Cup hopes after picking up the injury in a 6-1 friendly thrashing of Hungary on Saturday, coach Bert van Marwijk said.
He will not travel to South Africa with the rest of the party but has not been ruled out for the World Cup finals which start on Friday (June 11).
"He made an unnatural move and then sustained the hamstring injury," coach Bert van Marwijk said.
"He will not join us tonight on our way to South Africa as he will undergo a scan tomorrow as it was no use to do it now."
Van Marwijk said he had feared injuries from the Netherlands' final warmup match but had started to relax until second-half substitute Robben hobbled off in obvious pain two minutes from time.
"My first thought was this can't be true, the end the final warmup match, everyone is fit and then this," he added.
"We now have to wait. I can replace Robben until 24 hours before our opening match but as long as there is hope he recovers in time he will stay in the squad."
Robben has been in glittering form for his club Bayern Munich this season helping them reach the Champions League final against eventual winners Inter Milan in Madrid last month.
Until Robben's injury scare, prospects ahead of the June 11-July 11 finals only looked bright for the Dutch who had surprisingly conceded a sixth minute lead to the Hungarians through Balazs Dzsudzsak.
Good work from Dirk Kuyt earned Robin van Persie a 21st minute equaliser and Netherlands really hit their stride after the break with second-half sub Robben and Wesley Sneijder, Mark van Bommel and Eljero Elia all on target.
The Dutch open their campaign on June 14 against Denmark, with Japan and Cameroon as their other opponents in Group E.
Earlier on Saturday, Nigeria lost key midfielder John Obi Mikel through a severely bruised ankle and on Friday England skipper Rio Ferdinand was ruled out with a knee ligament injury.
The Ivory Coast skipper Didier Drogba also looks likely to miss out with a broken bone in his arm.
