Michael O'Neill's men had been made to work for victory in San Marino on Saturday (AEST), but a 3-0 success ensured they would need just a point from this fixture to pull clear of their opponents.
The hosts earned their fifth straight triumph as West Brom duo Jonny Evans and Chris Brunt - the former the subject of heavy interest from Manchester City during the transfer window - found the net to ensure that a first World Cup finals since 1986 remains a real possibility.
Czech Republic had dropped to fourth place before kick-off with Azerbaijan's thrashing of San Marino and their slim hopes of qualification were completely wiped out before half-time through Evans and Brunt, the pair typically profiting from set-pieces.
After cruising through the second half, Northern Ireland retain a remote hope of claiming top spot ahead of Germany, but a play-off place is the realistic target - and only a spectacular collapse would see O'Neill's side fall short as Europe's worst second-placed side.
Although tough tests against the world champions and Norway are to come, Northern Ireland, with seven clean sheets from eight and unbeaten in competitive home internationals since September 2013, will firmly believe they can reach next year's tournament in Russia.
Exactly a year on from a hard-fought goalless draw in Prague, the home side made a bright start and Josh Magennis - twice a scorer against San Marino - nodded an early corner wide.
The visitors soon settled but, faced with a defence as good as any in this campaign, they merely mustered a spell of harmless possession before the opener arrived at the other end.
With a Brunt corner from the right only half-cleared and Theodor Gebre Selassie slow to push up, Oliver Norwood's looping header behind the Czech back line found Evans onside, the defender throwing himself backwards to nod beyond Tomas Vaclik.
A second goal arrived just before half-time as Brunt swept a superb 25-yard free-kick around the visiting wall and into the bottom-left corner.
The same pattern of play continued after the restart and Aaron Hughes made a vital interception as a high ball threatened to finally bounce Czech Republic's way in the penalty area.
A series of attacking changes followed from the visitors, their pressure leading to a half-chance that Marek Suchy scuffed into Michael McGovern's grateful arms.
But a capacity Windsor Park crowd roared Northern Ireland over the finish line, with a first ever win against Czech Republic representing a huge step towards the play-offs.