King has not played for Spurs since their 1-1 draw against Chelsea on December 22 as he has struggled to return to full fitness because of his chronic knee troubles.
It was hoped that the 31-year-old, who played just nine games last season, would return at centre-back for Wednesday's huge Barclays Premier League game against Everton.
King pulled up in training on Friday holding his hamstring, however, and manager Harry Redknapp fears the defender has suffered a tear.
The news comes at the worst possible time for Redknapp, who only has Younes Kaboul, wantaway defender Sebastien Bassong and Michael Dawson, who has just returned from a four-month Achilles injury lay-off, available at the back.
"Ledley did his hamstring on Friday," Redknapp said.
"He had a little jog and tore his hamstring, we think. He has gone for a scan. We don't have the results yet but he only went for a little easy jog and his hamstring went."
Should King be ruled out for a long time, Redknapp may be forced to step up his bid to bring in a defender, with Blackburn's Christopher Samba thought to be high up on his shortlist.
"I have not got many options," Redknapp said.
"I only have Dawson, Bassong and Kaboul. No-one else."
Redknapp rested the vast majority of his first team for Saturday's FA Cup third-round win over Cheltenham, giving the likes of Roman Pavlyuchenko, Steven Pienaar and Giovani Dos Santos a rare chance to impress.
They obliged, putting on a fluent display against the npower League Two side that saw them run out 3-0 winners thanks to goals from Jermain Defoe, Pavlyuchenko and Dos Santos.
Dos Santos would have been credited with the first goal had Defoe not stolen in and tapped home the Mexican's goal-bound lob on the goal-line.
Dos Santos has scored just three goals in his four-year stint at the club - a time which has been punctuated by loans to Racing Santander, Galatasaray and Ipswich.
Redknapp has previously said that he thinks the Mexican's prospects have been hindered by his supposed love of the London party scene, and the Spurs boss expects him to leave White Hart Lane this month despite his exploits on Saturday.
"Gio is a boy with an amazing talent but he has to work harder if he wants to be a top player," Redknapp said.
"You can't change some people.
"Jake Livermore played at right-back on Saturday. He has never played there before in his life but he went in there and gave me everything.
"He (Livermore) comes in every day and trains like a lunatic. He loves it, so does Michael Dawson and Danny Rose too. Most of them are the same but you will always get one or two who will get fed up.
"They think that they're not getting a chance and they want to leave and I think Gio has got a bit like that.
"He is fed up here. He probably finds that this is not the ideal place for him at this time."
Part of Redknapp's problem with Dos Santos is that he did not want to move to Udinese last summer despite the Italian side offering £13million for his services.
"Udinese came in in the summer and offered us something like £13million for him and the president came over and spoke to him but he didn't want to go," Redknapp added.
"It was good money and Udinese are a good club. They were in the Champions League at the time."