The 20-year-year-old suffered a knee injury in his side's defeat to Feyenoord and has been ruled out for the remainder of the campaign as well as the tournament in Brazil
The 20-year-old was forced off in the first half of his side's 2-0 defeat to Feyenoord on Sunday and faces "several months" out after damaging "the inner tube of his knee".
Willems has 11 caps to his name, with his most recent coming in the 2-2 draw against Japan in November last year, but will not be available to Louis van Gaal for the competition in Brazil.
Following tests on Monday, PSV's medical staff confirmed that he will play no further part this season and will instead focus on the next campaign.
"Jetro Willems is no longer in action for PSV this season," read a statement on their official website. "The left-back has damage to the inner tube of his knee and is therefore out for several months.
"Willems now focuses on preparing for the new season."
The Netherlands, who will also be without Roma midfielder Kevin Strootman due to a knee injury, start their World Cup campaign against reigning champions Spain on June 13 before they take on Australia and Chile in Group B.