The Spurs full-back sustained an ankle injury during his side's 5-2 Premier League loss to Arsenal on Sunday.
Walker joins Wayne Rooney, Tom Cleverley and Darren Bent in withdrawing from Stuart Pearce’s squad, his first as caretaker manager following the resignation of Fabio Capello earlier this month.
Rooney and Cleverley both missed Manchester United’s 2-1 win over Norwich at the weekend with a throat infection and an ankle injury respectively.
Meanwhile, Bent withdrew after injuring his ankle in Aston Villa’s 0-0 draw with Wigan.
Pearce will not call any replacements up to the squad meaning Fraizer Campbell is likely to make his England debut, with only Daniel Sturridge and Danny Welbeck remaining as forward options.
Micah Richards may hope to replace Walker at right-back after returning to the international scene following a frustrating failure to make the England team during Capello's reign.
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