Better Together chair Alistair Darling addressed a crunch meeting of delegates at the Union of Shop, Distributive and Allied Workers (Usdaw) Scottish conference, but no-one from Yes Scotland was represented.
The union's statement that 82% of delegates voted against independence has also been queried after it was claimed that a proportion of attendees did not vote.
Usdaw has over 400,000 members across the UK and is affiliated to the Labour Party.
In 2012, the union's annual delegate meeting in Blackpool agreed to back the Better Together campaign at a UK level.
Its in-house magazine then asked for members to send their views on independence to Usdaw's campaigns officer Karen Whitefield, a former Labour MSP and now the party's prospective Westminster candidate for Falkirk.
Delegates from Scotland were then given an opportunity to vote on independence at Usdaw's Scottish divisional conference in Dundee earlier this month.
Darling made a speech criticising independence before taking questions from delegates, but no-one from Yes Scotland appeared alongside him to make the argument in favour of independence.
After the vote, Usdaw released a statement saying the union had "overwhelmingly supported keeping Scotland in the United Kingdom, with 82% voting against independence".
But the union has now been criticised over the format of the event.
One shop steward said a number of delegates had not voted either way, and the 82% figure related to the smaller number who had participated in the ballot.
The union official said: "The union at a UK level backed Better Together and then asked delegates in Scotland to vote on independence without giving them the chance to hear both sides of the argument.
"There was little engagement with the membership.
"It has been a completely cynical exercise and a stitch-up."
Usdaw has donated over £12 million to the Labour Party since 2001 and over £25,000 to the Scottish party and local constituencies since last year.
Colin Fox, co-convener of the pro-independence Scottish Socialist Party, said: "It looks bad that a London-based union has not even allowed a Yes speaker to put the independence case to its delegates.
"Does Usdaw even know how to spell democracy?"
The Sunday Herald contacted Usdaw's press office and asked to speak to an official but no comment was forthcoming.
A spokesperson for Yes Scotland said: "It's disappointing that we weren't invited to put forward Yes Scotland's case.
"Had we been, we would have told delegates about the gains of independence for workers including how their rights would be enshrined in an independent Scotland's written constitution and how the Living Wage could be extended further across the public and private sectors."
The row comes after the UK executive of the Communication Workers Union backed a No vote without staging a special conference of Scottish members.
The GMB union was also criticised for the internal process that led to its No endorsement.