In the end, Ronnie Moore's revamped side could have few complaints - striker Edrissa Sonko's third and final embarrassing miss summed up his night.
In a thoroughly entertaining tie, Grimsby rode their luck before a controlled, skilful and at times outstanding display saw them cruise through to round two.
The Carling Cup is a competition they have graced with good runs - but not in recent seasons.
But all this seemed a long way ahead when defender Andy Taylor and skipper Antony Kay both hit the woodwork either side of Grimsby's 20th minute opener.
That came when striker Martin Butler served up a chance for midfielder James Hunt, who motored into the box to loop a header past keeper John Achterberg.
The last time the sides met, Tranmere's 2-1 win condemned Grimsby to the bottom flight and they are still there five seasons later.
But Grimsby never fear teams from higher divisions on a cup night at Blundell Park.
On Saturday, Grimsby's new look line-up were stretched in a goalless draw with Rochdale, but Tranmere, who were beaten 3-1 in their League One opener at Swindon, were expected to provide even sterner opposition.
But manager Moore, such a fine finisher in his playing days, could not believe the chances his side squandered.
Defensively they were abject at times, but Grimsby's crucial second goal on 54 minutes was cruel on Tranmere.
Even so, much credit goes to midfielder Chris Llewellyn whose teasing cross was dispatched past his own keeper by defender Ben Chorley.
After that there was no way back for the boys from Birkenhead.