An ongoing knee problem has got progressively worse, resulting in his absence against Perpignan on Saturday and subsequent withdrawal from Eddie Jones' 36-man training squad, his place taken by Elliot Daly.
May has been an ever-present under Jones since 2017 and is an influential member of the backline, creating a highlights reel of spectacular tries, but he now faces a race against time to involved in England's title quest.
The February 26 showdown with Wales at Twickenham is the earliest he will be back, but the country's second most prolific try-scorer behind Rory Underwood could also be looking at a lengthier lay-off.
"Jonny's seeing some people this week, but it will definitely be a good couple of weeks. He's seeing the best of the best," Gloucester head coach George Skivington said.
"He's had a niggle for a little while and it kept him out of one game a few weeks ago. It's just been chipping away and it got to a point where it wasn't great, so we decided to get it investigated.
"The hope is that he will still play in the Six Nations. If he can back involved in the Six Nations then great. It's unfortunate for Jonny because we'd like to see him playing for England.”
Owen Farrell will also miss the tournament opener at Murrayfield because of an ankle issue and his place in the squad has been taken by George Ford.