The former Burnley man, who has already had two spells out of action this season due to a hamstring complaint, suffered his latest injury this morning at home as he walked downstairs.
Little reported that he suffered a tightening of the muscle - and Steve Coppell decided that it was best that he missed this afternoon's game, not wanting to risk aggravating the winger's injury.
Although the complaint is only minor, Little could potentially face a couple of weeks out of action recovering.
Speaking after today's 2-1 win over Cardiff, Coppell admitted that he thought his side did well without Little, who has played eleven games so far this season.
"We had the flexibility to cope with that and Hughesy played well and was unlucky not to score just before he came off," said the Royals boss, speaking to the official site.
"We were a little bit unsettled with Glen not being fit. He tweaked his hamstring running up some stairs this morning so we had to rearrange just after one o'clock."
With both Dean Morgan and Andy Hughes in superb form, the Royals look unlikely to miss influential wing man Little too much.