Real confirmed in midweek that Militao felt a twinge in a challenge shortly before halftime in Tuesday's win against Alaves and signalled straight away to be replaced.
While Real coach Alvaro Arbeloa initially struck an optimistic tone after the game, an MRI scan later revealed a muscle injury to the hamstring in Militao's left leg.
Spanish radio COPE first reported it was a recurrence of the injury he sustained in December, which could require surgery and rule him out of the World Cup, which starts in less than 50 days. Reuters has confirmed that report with several sources.
The 28-year-old now faces an anxious wait. Sources told Reuters a final decision will be made and announced on Monday, when Militao undergoes a new series of examinations with Real's doctors to determine the next steps.
The latest blow marks another setback for the Brazilian centre back, who has endured a succession of injuries in recent seasons, including ACL tears in back-to-back years.