Warren Joyce's holders lost the grip on the trophy despite leading for much of the game through a Joe Rothwell spot-kick as Zach Clough popped up with a late equaliser for the dogged Trotters.
Tom Thorpe returned as captain after an ankle injury cut short his loan at Birmingham City and his goal-saving challenge on Andy Kellett proved his fitness. Tame efforts were then provided by Clough, Rob Hall and United's Andreas Pereira before a dramatic spell unfolded.
Guillermo Varela appeared to be tripped inside the box but nothing was given and Wanderers broke with Kellett's cross appearing to strike Thorpe's hand. However, no penalty was awarded again until, moments later, Rothwell was felled moving away from goal by Hall on the edge of the area.
Rothwell took on the responsibility himself to send Arran Lee-Barrett the wrong way from the spot and open the scoring. James Wilson fired a ferocious drive just wide on the turn after good work by the tricky Andreas Pereira on 34 minutes before the visitors enjoyed a period of domination.
Joel Castro Pereira made a couple of routine stops before pulling off an amazing reflex save to keep out Clough from close range after a Hall free-kick was headed back across by goal by James Kelly. Bolton appealed the ball had crossed the line but the keeper's heroics maintained the lead heading into the break.
The second half was one of few chances as the play became rather scrappy on a difficult surface at Altrincham's Moss Lane. Ben Pearson produced a wayward shot over the bar before setting up