With four Under-16s in the squad and two Under-18 development players starting, the Robins started brightly with an early goal from in-form Lewis Thompson.
Good play by Jamie Goodwin saw his cross-shot go across the face of the goal, with Thompson following in to tap-in from close range.
Cheltenham continued to play well in what was a well-contested, fair game. Colchester had a few chances but some good defensive headers and saves from Bojan Tetek kept the Robins in the lead.
In the 35th minute, after some fine individual play, Thompson had a right-footed shot from outside the area which hit the bar and bounced down; it looked to have crossed the line but the officials waved play on.
Left-back Dan Okojie, just returning from injury, was substituted at half time with Under-16 right-back Taylor Cordell coming on, with Danny Reynolds moving out of centre-back and going to left-back.
Colchester started the second half brightly and were rewarded when a cross from the right was headed in by Louis Dunne at the back post to level the scores in the 52nd minute.
A short period of sustained pressure from Colchester then saw Callum Craddock tackle Joel Atkin in the box on 55 minutes, leading the referee to give a penalty. Atkin then proceeded to take the penalty, slotting the ball to Tetek’s left.
Jordan Lymn replaced Jamie Goodwin and with 15 minutes to play, Under-16 Luke Thomas replaced Reece Daly and the Robins kicked on in search of an equaliser.
The last 15 minutes saw Cheltenham camped in Colchester’s half with James McCarthy starting attacks down both the left and right flanks.
Both Thompson and development squad player Adam Page had good chances to score but their efforts were saved.
This was a brave effort from Cheltenham who will be disappointed to not have got a result out of the game with the chances they created, but they should be proud of their efforts to have got to round two of the FA Youth Cup. Colchester will play Ipswich Town at home in the third round.
Cheltenham Town: Tetek, Craddock, Okojie (Cordell 46), Daly (Thomas 75), Rowe, D Reynolds, Goodwin (Lymn 55), McCarthy, Dinsmore, Thompson, Page. Other subs: Nutting, Mendes.