Butland was due to feature for the Potters' Under-23 side in Friday's Premier League Cup tie at Reading.
But he pulled out after feeling pain in the warm-up and was sent for a new MRI scan on Saturday.
"It's a little bit of a concern, I have to say," said Hughes.
Speaking after Saturday's 2-0 Premier League home win over Burnley, Hughes added: "I haven't had an update in terms of where we think he is with it but he's gone for scans.
"He got to the warm-up and felt discomfort in the ankle, the same situation that we found ourselves before the beginning of the season, unfortunately.
"Not to the same extent, but in the same area, so we need to get to the bottom of it. Hopefully we can settle it down again.
"If that's not the case then we'll have to try and do something more radical, maybe, but we'll try and avoid that if we can."
The 23-year-old, capped four times by his country, missed Euro 2016 after being injured in England's 3-2 win over Germany in Berlin in March. He was then kept sidelined for two further months after more surgery in September.
It is more than three months before England's next fixture - a friendly against Germany in Dortmund on 22 March. But Hughes has already hinted that Butland could face a battle just to get back in the Stoke team.
On-loan Derby County goalkeeper Lee Grant has so far conceded just five goals in eight matches since replacing Shay Given in September to help the rejuvenated Potters climb to ninth in the Premiership table.
Stoke City boss Mark Hughes is fearful that England goalkeeper Jack Butland may have suffered a further setback after eight months out following surgery to repair a fractured ankle.