The home side overcame Forest Green Rovers in the last round to progress, while Chelsea Under-21s advanced to the round of 16 courtesy of their 4-2 success over Peterborough United.
Match preview
Cheltenham finished second in Southern Section Group C, collecting six points from their three matches, to advance to the last-32 stage of the competition, where they beat Forest Green Rovers on penalties.
Chelsea Under-21s are now standing between the League One outfit and a spot in the quarter-finals of the Southern Section, and they will enter this match having won three of their last four games in all competitions.
Wade Elliott's side did suffer a 1-0 loss to Forest Green Rovers in the league on Saturday, though, which has left them in 17th position in the table, boasting 25 points from their 20 matches.
Cheltenham have already been knocked out of the FA Cup this season, suffering a shock 2-1 loss to Alvechurch last month, so the EFL Trophy has taken on added importance this term.
League One will continue to take priority for the club, though, and head coach Elliott will certainly have one than one eye on the key league encounter with Lincoln City next Saturday.
Chelsea Under-21s, meanwhile, are likely to be missing some important players for this match, with many of the youngsters involved in the first-team's mid-season trip to Abu Dhabi.
As a result, Mark Robinson's side will be far from full strength, but the youthful outfit will still fancy their chances of causing plenty of problems for their League One opponents.
Chelsea Under-21s finished top of Southern Section Group G with six points to book their spot in the round of 32, where they beat Peterborough 4-2 to advance to this round.
Malik Mothersille registered twice for the Blues against Peterborough, while Cesare Casadei and Ben Elliott were also on the scoresheet during a strong attacking performance.
Cheltenham Town EFL Trophy form:
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W
Cheltenham Town form (all competitions):
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D
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Chelsea Under-21s EFL Trophy form:
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W
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Chelsea Under-21s form (all competitions):
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Team News
Cheltenham head coach Elliott could select the same XI that took to the field against Forest Green Rovers in the last round of the competition.
There will be changes from the side that started the league meetings between the two teams on Saturday, with Charlie Brown and Charles Raglan among those expected to come into the XI.
However, Dan N'Lundulu should retain his spot in the final third of the field for the League One outfit.
As for Chelsea Under-21s, Eddie Beach, Bashir Humphreys, Alfie Gilchrist, Dion Rankine, Lewis Hall, Casadei and Mothersille are among the players involved with the first team in Abu Dhabi.
As a result, there are likely to be some key absences for this match, but there is a chance that some of the youngsters with the first team could still be involved in this contest, with the players set to travel back to London after taking on Aston Villa in a friendly.
Cheltenham Town possible starting lineup:
Southwood; Raglan, Taylor, Freestone; Jackson, Bonds, Sercombe, Hutchinson; Broom; Brown, N'Lundulu
Chelsea Under-21s possible starting lineup:
Beach; Brooking, Gilchrist, Williams; Abu, Elliott, Thomas, Sturge; Burstow, Mothersille, Castledine
We say: Cheltenham Town 2-1 Chelsea Under-21s
The strength of Chelsea Under-21s for this match is yet to be confirmed, but it will be a tough game either way for the Blues against a decent League One outfit, and we are tipping Cheltenham to edge this contest to advance to the next round of the competition.