The 1921 painting, titled Portrait of Mary Lushington Bernard D'Oyly, was never included in the artist's catalogue raisonne but has been in the sitter's family in Devon, England, for decades.
They had always believed it was a real Roberts but it wasn't properly authenticated until earlier this year, Anne Gerritsen from auctioneers Dreweatts said.
It is rare to unearth a previously unknown work by an artist as important as Roberts, Gerritsen said.
"Some of these works can be in family collections for centuries ... so if they never come to market then people might not be aware of them at all," she told AAP.
Roberts was born in the United Kingdom in 1856 and became one of the founding members of the Heidelberg School, which was at the forefront of the development of Australian Impressionism.
While the portrait has only recently come to light, intriguing clues have pointed to its existence for more than a century.
Previous research on the artist's life has noted he was in the village of Combe Raleigh, near Honiton in Devon, in the third week of August 1921 to paint a portrait of a Mrs B D'O.
In a letter to his wife held by the State Library of NSW, Roberts included a sketch of the portrait and noted "the colour is a good pale blue on Gold B'ground".
Roberts wrote the painting had not been progressing happily but he had since "got hold of it" and it was going well.
A former senior curator of the National Gallery of Australia, Dr Mary Eagle, was able to link the portrait with the letter and sketch, according to Gerritsen.
"She immediately connected the dots and made that link, so that was brilliant," she said.
The portrait had been owned by one of the sitter's five children, Marjorie Churchill, and passed down to her descendants.
Roberts also wrote to his wife that Mary Lushington Bernard D'Oyly was "very spring like & lace & pearls", although the proponent of plein air painting said he found working indoors restrictive.
"There is much difficulty in working in an ordinary room - one gets jammed up, the light deceives one - when the place is not spacious & you are right against the window," the letter reads.
Portraits were Roberts' bread and butter, although his landscapes have always been more popular on the secondary market.
The painting will be offered at auction in the UK on Wednesday in a sale of Old Master paintings & European Art.
The conservative auction estimate is about Stg 10,000 to 15,000 ($A18,000 to $27,500).
In 2017, another Tom Roberts initially thought to be a fake was authenticated via BBC TV program Fake or Fortune.
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