Both sides are kicking off their campaign this week, ultimately fighting it out for a top-two finish in a group also featuring Barrow and Bolton Wanderers.
Match preview
Having made an unbeaten start to the League Two season, Fleetwood Town travelled south to the capital in hope of extending that run on Saturday, but instead Charlie Adam's side were consigned to their first defeat.
Fleetwood came away from London empty-handed following a 1-0 loss to AFC Wimbledon, leaving them with a tally of five points from their first four matches.
After enduring relegation from League One, an immediate return to England's third tier is the Lancashire club's sole objective for their 2024-25 campaign, albeit knockout football has proven a welcome distraction.
Goals have not flowed freely for the Fishermen, but they did score twice in both of their EFL Cup ties during August, in which they saw off West Bromwich Albion and then Rotherham United.
Ryan Graydon found the net three times across those two matches against higher-level opponents, so the Irish striker may fancy his chances of exploiting an inexperienced defence on Tuesday evening.
Last time out, Aston Villa Under-21s were picked off by Arsenal's latest crop of talented youngsters, suffering a 4-1 away defeat on Friday.
Head coach Josep Gombau had previously helped his team take four points from their first two games of the Premier League 2 season, following a win over Derby County with a 1-1 draw at Leeds United.
Last term, Villa finished 16th, which was just good enough to snatch a spot in the playoffs, but they were then eliminated in the last 16 after an extra-time defeat to eventual champions Tottenham Hotspur.
The Midlands side also finished rock-bottom of their EFL Trophy group in 2023, picking up just one point in the process, so they will hope to fare better this time around.
Fleetwood Town form (all competitions):
W
W
D
D
W
L
Aston Villa Under-21s form (all competitions):
Team News
Likely to make changes to his starting XI in midweek, Charlie Adam could select recently arrived Manchester United loanee Rhys Bennett in defence, after the 20-year-old centre-back appeared as a substitute on Saturday.
Kayden Hughes also made his league debut for Fleetwood at the weekend, and Huddersfield Town forward Kian Harratt has just joined on a season-long loan.
Ex-Arsenal defender Zech Medley will not be available, though, after undergoing surgery on an Achilles injury.
Back-up goalkeeper Jay Lynch started in both of Fleetwood's EFL Cup ties, but Aston Villa Under-21s will stick with regular number one Sam Lewis, as they name their strongest XI.
Ben Broggio is the visitors' top scorer this season with two goals, while Scottish striker Rory Wilson should start up front after scoring a penalty against Arsenal. Kobei Moore - son of former Villa player Stefan Moore - could also feature in the final third.
Fleetwood Town possible starting lineup:
Lynch; Wiredu, Holgate, Bennett; Johnston, Bonds, Mayor, Virtue, Patterson; Graydon, Harratt
Aston Villa Under-21s possible starting lineup:
Lewis; Katsukunya, Smith, Amundsen-Day, Patterson; Pierre, Alcock, Borland, Broggio; Wilson, Moore