The Wolves stopper suffered a serious head knock in a match against Liverpool last week and reportedly suffered a concussion.
He was initially called up to the Portugal squad but has eventually had to withdraw after he was not deemed healthy enough yet to feature following the blow to the head.
Jose Sa of Olympiacos is his replacement in the squad, though Anthony Lopes could be given the nod from the start.
Portugal have a raft of experience within their defensive ranks, so Pep might actually be given a start despite his advancing years due to his outstanding recent performances for Porto, particularly in the Champions League.
Jose Fonte or Domingos Duarte are the other options to partner Ruben Dias in the centre of defence.
Joao Cancelo will start at right-back, with Raphael Guerreiro likely to be on the left.
Over the last handful of games they have played a 4-2-3-1 on one occasion and a 4-3-3 the other couple of times.
Should Fernando Santos opt for a 4-2-3-1 formation, Ruben Neves could partner Danilo Pereira in the two.
Cristiano Ronaldo will start as the central striker.