Wilkinson was forced out of training on Tuesday and medical examinations found he was suffering "an acute injury to his right adductor".
Wilkinson tore his adductor muscle in January, but had returned to full training and was considered fit enough to be named on the bench for Newcastle last weekend, though he did not feature in the 9-6 win over London Irish.
The latest injury is unrelated to the operation Wilkinson had on a hernia last November.
Falcons director of rugby, Rob Andrew, said: "This is an unfortunate setback, and is in the hands of the medics, who will continually monitor the situation."
Wilkinson must feel like he is a cursed man. He has not played for England since the 2003 World Cup final and his career has been punctuated by injuries for the last two and a half years.
Wilkinson underwent shoulder surgery after the World Cup and after returning to action he suffered injuries to his bicep and two knee ligament problems during the 2004/05 season.
Wilkinson recovered in time to make the Lions tour but a lack of first team rugby affected his form.
The 26-year-old had an appendix removed after falling ill on Newcastle's summer tour of Japan before going under the knife again in November as surgeons operated on a hernia.
Newcastle and the medical team are confident that work is still holding strong and that this latest set-back is unrelated.
Newcastle and England have been extremely careful not to over-burden Wilkinson or rush him back before he is ready.
England's situation has been helped by the development of Charlie Hodgson, who is arguably the best fly-half on current form in the RBS 6 Nations.