Ashton completed the 90 minutes, with the Hammers having already used their three substitutes, but he looked laboured and his worst fears were confirmed after the game.
It is another massive setback for Ashton, who had battled back from a broken ankle which he suffered in training ahead of England's game with Greece 14 months ago - the first of Steve McClaren's reign.
McClaren was also at Villa Park to witness this latest blow for Ashton, whose side were beaten 1-0 by a first-half free-kick from Craig Gardner.
West Ham manager Alan Curbishley said: "Dean has hurt his medial knee ligament. You could see he was labouring and couldn't really run about.
"I would think he is very doubtful for England. He couldn't really run near the end so I imagine that is it for him.
"He swung his leg to clear the ball and has caught his boot on somebody and tweaked his medial knee ligament.
"It's a big blow for England but it's also a big blow for us as we have injuries aplenty up front.
"It all depends how badly he has hurt his ligaments. We will have to wait and see but I would say things are looking a bit tight for the next two weeks.
"You feel sorry for the lad because he has had his fair share of bad luck and has worked ever so hard to get himself back fit and playing."