The France international played the full 90 in the first leg last week, but he lasted just 45 minutes against Osasuna at the weekend before being substituted with discomfort in his leg.
Now, Los Blancos have revealed that Varane has indeed suffered an injury to his right abductor muscle, and as a result he will not be available for the trip to Stamford Bridge.
Zinedine Zidane should however be boosted by the returns of Ferland Mendy and Sergio Ramos, though, with both men returning to team training on Monday morning.
Varane joins Lucas Vazquez and Dani Carvajal on the sidelines, but fellow defender Marcelo should be cleared to play despite being called up for polling duty.
Real head to the English capital after a 1-1 draw in the first leg of their clash with Chelsea, during which Christian Pulisic broke the deadlock before Karim Benzema's 71st Champions League goal levelled the tie.