Nathan Blake made amends for his red card against Rotherham with a first-half winner at Blundell Park which moved Wolves closer to the Division One play-offs.
Blake was dismissed for an apparent elbow on Millers defender Shaun Barker during Saturday's goalless draw at Molineux, but the former Blackburn striker responded with a clinical finish against Grimsby to give his side three crucial points.
The result moved Wanderers up to fifth in the table but, more importantly, gives the club a six-point cushion over play-off hopefuls Norwich and Ipswich with just five games remaining.
Wolves' defeat by Grimsby towards the end of last season had started the poor run which saw the Molineux club miss out on automatic promotion to the Premiership, and Dave Jones' players looked nervous from the kick off on Tuesday.
Goalkeeper Matt Murray was tested after just two minutes, reacting quickly to stop a 25-yard effort from Richard Hughes.
However, the visitors began to settle and soon threatened through Blake, who flashed a shot wide from the edge of the box.
And with 12 minutes gone, the striker gave Wolves an early lead, running on to Kenny Miller's neat pass and driving a low strike past keeper Danny Coyne and into the corner of the net.
Veteran midfielder Paul Ince also threatened after 28 minutes, but headed wide, before a foul on Colin Cameron earned midfielder Richard Hughes a caution.
Murray blocked George Santos' header and Hughes fired wide as the Mariners fought back, but it was Wolves who came closest to forcing a second goal when Coyne kept out Miller's header on the stroke of half-time.
The Grimsby keeper eclipsed that stop within three minutes of the restart when he dived full-stretch to tip an 18-yard effort from Blake over the crossbar.
It was a rare opportunity during a quiet start to the second period, and Wolves had to wait until the 79th minute for another chance to wrap up victory.
Kevin Cooper was given space on the edge of the box, but his shot lacked accuracy, leaving the visitors to survive a tense closing spell.
Grimsby: Coyne, McDermott, Ford, Santos, Gallimore, Campbell, Bolder, Groves, Richard Hughes, Keane, Mansaram.
Subs: Allaway, Livingstone, Chettle, Boulding, Cooke.
Wolverhampton: Murray, Irwin, Lescott, Butler, Naylor, Ndah, Cameron, Ince, Kennedy, Miller, Blake.
Subs: Oakes, Rae,
Sturridge, Edworthy, Cooper.
Referee: E Wolstenholme (Lancashire).