Parent club Oxford United do not want the 19-year-old cup-tied and turned down a request from the Blues for him to be allowed to play.
Oxford have a number of players out on loan but only Michael Collins, who is with York City, will be allowed to feature in the FA Cup.
Roberts would have been a doubt for the tie anyway having injured his hamstring in the 2-1 victory against FC Halifax Town on Saturday .
In his first appearance since the death of his 16-year-old brother Ben a fortnight ago , Roberts was Chester's most dangerous player and scored the opening goal on the stroke of half-time.
But his game ended eight minutes into the second period when he went down inured and had to be replaced.
Chester are waiting for news on the extent of the injury to Roberts but manager Steve Burr knew before the Halifax game he would miss the trip to Moss Lane after talks with his opposite number at Oxford.
Burr said: "I spoke to Michael Appleton on Thursday last week. I didn't expect James to be available for the cup and they don't want him cup-tied but that will give somebody else an opportunity to come into the team."
He made an eye-catching debut in the 3-2 defeat of rivals Wrexham but that evening his younger brother was killed in a collision near Aylesbury.
Roberts was given compassionate leave but indicated he was ready to return for the game with Halifax .
He took to Twitter to express his gratitude for the support he received from Chester fans.