The Spireites have already guaranteed progression to the next phase of the competition, while their visitors have nothing to play for, having already been eliminated with a game to spare.
Match preview
Chesterfield boss Paul Cook has every reason to smile regarding his side's EFL Trophy exploits, as Tuesday's hosts are guaranteed to finish in the top two of the standings no matter what transpires on Tuesday evening.
The Spireites - winners of the tournament in 2012 and runners-up two years later - kicked off Group G proceedings with a 1-1 draw and subsequent penalty-shootout win over Manchester City's Under-21s, earning a bonus point in the process, before a strike from Connor Cook - the manager's son - sank Lincoln City in September.
With five points on the board from two outings, Chesterfield are sitting pretty at the summit - two points clear of Lincoln and Man City, who play each other this week - and even a draw and penalty success versus Grimsby would be enough to guarantee top spot.
The hosts' knockout successes have not just been limited to the EFL Trophy - they overcame Horsham 3-1 in the first round of the FA Cup on November 2 - although their nine-game unbeaten run in all tournaments came to an end with a crushing 3-0 home loss to Accrington Stanley on Saturday.
Nevertheless, Cook's team are still making a case for promotion in ninth place in the League Two table, and they hold the honour of being the top scorers in the fourth tier of English football, netting an unrivalled 28 goals in 15 matches thus far.
The side with the best offensive record in the division now face off against one of League Two's most porous defensive outfits - only three sides have let in more than Grimsby's 25 efforts thus far - and the visitors are only fighting for their own pride this week.
The Mariners have not been carved open at will in the EFL Trophy, but failing to hang onto a one-goal lead in an opening 2-1 loss to Lincoln was a devastating setback, before they also drew 1-1 with Man City's academy prospects and lost the subsequent penalty shootout.
Only boasting the one point from their two fixtures, Grimsby cannot force their way into knockout contention - one of Lincoln or Man City will have at least five points on the board by the close of play - so the Mariners will soon be able to focus solely on League Two matters.
David Artell's men have experienced another dose of knockout heartache recently, suffering a shock 1-0 FA Cup first-round loss at home to Wealdstone, although they beat AFC Wimbledon by the same scoreline on Saturday to cement seventh place in the League Two rankings.
Chesterfield and Grimsby have already faced off once in fourth-tier action this season, and it was the Spireites who flexed their muscles in a 2-1 home victory in September, ending a four-match winless run against the Mariners stretching back to 2009.
Chesterfield EFL Trophy form:
W
W
Chesterfield form (all competitions):
D
W
D
W
W
L
Grimsby Town EFL Trophy form:
L
L
Grimsby Town form (all competitions):
L
W
L
L
L
W
Team News
Chesterfield have lost two players to international call-ups this month, as 19-year-old forward Liam Jessop will be representing Gibraltar, while right-back Devan Tanton has been summoned to join Colombia's Under-23 team - a problem for the hosts with Vontae Daley-Campbell nursing a hamstring concern.
In addition, Everton-owned midfielder Jenson Metcalfe is cup-tied - he represented the Toffees' Under-21s in the opening round of the competition - although he was an unexpected absentee from the squad to face Accrington Stanley, as was Harvey Araujo.
Whether the latter will be fit to face Grimsby is unclear, but Ryan Boot should be given the nod in goal after Max Thompson hurt his ankle in the warm-up to Saturday's defeat.
As for Grimsby, the Mariners do not have to worry about any players leaving for national team camps this month, nor does Artell have any troops cup-tied for Tuesday's contest.
However, on-loan Wrexham midfielder Jordan Davies is currently missing with the hamstring injury he picked up in August, although there is optimism that he will be back within a couple of weeks.
While the visitors performed valiantly at Wimbledon on Saturday, Artell should not shy away from making a few alterations as he prioritises their promotion charge.
Chesterfield possible starting lineup:
Boot; Colclough, Williams, Grimes, Gordon; Oldaker, Banks; Markanday, Mandeville, Jacobs; Madden
Grimsby Town possible starting lineup:
Auton; Cass, Tharme, McJannett, Hume; Ainley, McEachran; Svanthorsson, Luker, Barrington; Obikwu
We say: Chesterfield 2-0 Grimsby Town
As pitiful as Chesterfield's display against Accrington Stanley was at the weekend, Cook's men have otherwise proven to be near-unbeatable over the autumnal period and have already asserted their authority over Grimsby once this term.