Insiders believe the Public and Commercial Services Union (PCS) will come out in favour of a Yes vote following a meeting of branch delegates tomorrow.
Backing from the union, which represents 30,000 civil servants in Scotland, would come as a huge boost to the Yes campaign after a series of challenges to the SNP's currency and economic plans. No major union has so far endorsed independence.
Delegates are gathering in Glasgow after a consultation process in which workplace branches have been asked their views on independence.
They will vote on three propositions: whether the union should remain neutral in the debate or back a Yes or No vote.
Deputy First Minister Nicola Sturgeon and Labour MSP Neil Findlay, who chairs Holyrood's PCS group, are both due to address the gathering.
Lynn Henderson, the PCS Scottish Secretary, will speak in favour of the union remaining politically neutral.
However, a well-placed source said: "I would be shocked if the vote is for anything but a Yes position."
Earlier this week the union's East Kilbride Revenue & Customs branch, which represents 2250 workers at the town's tax office, voted in favour of backing Yes.
The 1000-strong Glasgow DWP branch also voted in favour of independence earlier this month.
Another union source said: "Listening to the debates within the branches, the final vote is going to be close between adopting a neutral position and supporting Yes."
The result of the vote is almost certain be endorsed by the UK-wide union at a conference later this year.
Most PCS members are employed by the UK Government - but insiders said many were disillusioned after years of spending cuts. Speculation about the PCS vote came after the STUC published its latest thinking on the independence debate in a new report, A Just Scotland.
STUC general secretary Grahame Smith said it would be up to the umbrella organisation's 40 affiliatated unions whether it remained neutral or backed either side in the campaign in the run-up to September 18.
A number of unions, including the GMB, ASLEF, Community and Usdaw, have backed a No vote but insiders believe the STUC is likely to remain neutral.
In its report, the STUC challenged the SNP to make a stronger case for a currency union with the rest of the UK.
It warned an independent Scotland would be in a weak negotiating position if it could not demonstrate that Alex Salmond's under-fire pound-sharing proposal was in the best interests of the rest of the UK as well as Scotland.
The issue also dominated clashes at Holyrood. New figures highlighted by Labour showed Scots firms would face money-changing costs of £1230 on average if the country left the UK and had to set up a currency, new figures suggest.
The calculations, by the Scottish Parliament's politically neutral information centre, showed transaction costs for Scottish businesses would be £400million per year.
The figure compares with a £500m cost - equal to £109 per business - to firms south of the Border trading in Scotland.
Scots Labour leader Johann Lamont yesterday described the costs as an "Alex Tax" which would be imposed if First Minister Alex Salmond won a Yes vote in the referendum.
She said that, proportionately, a new currency would result in higher transaction costs in Scotland than the rest of the UK.
But Mr Salmond hit back: "Our proposal is to share the pound and not have the transaction costs.
"The point that is being made by the Scottish Government is a reasonable one - I don't think English businesses will take kindly to be forced to pay the George or Johann Tax."
He repeated his threat that an independent Scotland would not accept a share of UK national debt, estimated at £130billion, unless his proposed currency deal was agreed.
Mr Salmond mounted a robust defence of his under-fire pound-sharing plan after a poll suggested Mr Osborne's decision to rule it out had boosted support for a Yes vote.
The Survation survey - the first since the Chancellor's intervention last week - showed the No campaign's lead narrowing to nine points, with 47% opposed to independence and 38% in favour. The previous comparable poll showed a 12 point lead for No.
But the poll also showed two-thirds of Scots wanted Mr Salmond to outline a "Plan B" for currency.