The painting, which hangs in the Scottish National Portrait Gallery on Queen Street in Edinburgh, has been attributed to Allan Ramsay (1713-1784), one of Scotland’s greatest artists.
But art consultant Robin Nicholson has claimed it was actually the work of a lesser-known artist Cosmo Alexander, reducing its value to under £200,000, less than a fifth of the price paid.
He said: "The painting is well executed, but not [a] Ramsay. It totally lacks the effervescent effect of light that became Ramsay's trademark."
The painting, which measures just 12in x 10in, was acquired by the portrait gallery (SNPG) in 2016 under the “Acceptance in Lieu” scheme in settlement of £1.1m of estate duty arising from the death of the 11th Earl of Wemyss in 1937.
The National Portrait Gallery archives in London recorded the painting as a copy of an engraving by Robert Strange.
Art historian Bendor Grosvenor said it was by Ramsay and his view was endorsed by Duncan Thomson, a former director of the SNPG who is an expert on Ramsay's work.
Announcing the acquisition in March 2016, the National Galleries of Scotland (NGS) described the picture as “a hugely significant portrait of Bonnie Prince Charlie by the greatest Scottish portrait painter of the 18th century”.
NGS said the portrait was thought to have been painted at Holyrood during Bonnie Prince Charlie’s short time in Edinburgh at the height of the 1745 Jacobite Rising.
Portrait gallery director Christopher Baker said at the time: “This meticulous and dashing portrait is a work of great historical resonance.”
And Edward Harley, chair of the Acceptance in Lieu panel, welcomed the return of the “iconic image of Bonnie Prince Charlie” to the city in which it was painted.
He said: “It now takes its fitting place as one of the highlights of the great collection of the Scottish National Portrait Gallery where it can be enjoyed by all. This is indeed a unique moment in Scottish history.”
However, Mr Nicholson, who curated the Drambuie Collection of Jacobite art, inspected the painting while it was hanging in East Lothian’s Gosford House as part of his research for his 2002 book Bonnie Prince Charlie and the Making of a Myth: A Study in Portraiture.
He told the Sunday Times he had "immediately recognised this was not an original", but a derivation of a work by Robert Strange.
"In my opinion, it is basically a copy of Strange's print,” he said. “If anything, I might attribute it to Cosmo Alexander and suggest it may have been painted in 1747.
"The purchase price for the SNPG was astronomical. If it is by Cosmo Alexander, it might be worth less than £200,000."
The National Galleries of Scotland said: "We welcome and support all serious research into the works in our collections.
"In the case of our portrait of Prince Charles Edward Stuart, acquired in 2016, we have undertaken extensive research into the painting's history and origins and, after reviewing all possible alternatives, are fully confident in its attribution to Allan Ramsay and dating to 1745."