The Fulham player is struggling with a groin injury he picked up in the final minutes of their 1-0 defeat to Newcastle on Saturday.
Irish boss Michael O'Neill could now be without one of his most experienced players, while Hibernian left-back Ryan McGivern has also emerged as a further injury concern.
McGivern was expected to replace suspended Burnley defender Daniel Lafferty but he missed training on Wednesday with a knock.
Blackpool's Craig Cathcart had already been forced out of the Portugal match and Tuesday's game with Luxembourg with a knee problem and O'Neill confirmed he will have to rejig his defence.
"Aaron has a groin problem from the last few minutes against Newcastle. We're in constant conversation and we left him an extra day with his club," O'Neill told Sky Sports News.
"He'll report today and see how he is, but he's an extreme doubt. Ryan is the same to be honest, an extreme doubt.
"We have Craig Cathcart out too so that limits us slightly. It'll be a different back four to what we had against Russia and we may have to look at players who wouldn't have full-back as their natural position.
"We have options and I don't know I'll call anyone in to be honest, but I'll assess it."
O'Neill had previously boosted his options by calling up versatile Aberdeen player Josh Magennis and Kilmarnock defender Rory McKeown.
But if Hughes and McGivern are forced to drop out he will face a serious selection headache ahead of the double-header.
O'Neill's side are fourth in Group F on six points, five behind third-placed Israel and eight adrift of leaders Portugal, having played a game less than the pacesetters.