The painting, originally by Leonardo da Vinci, was found in the Prado museum and is believed to be the earliest version of the portrait.
It is thought the new discovery was painted alongside the original, and so could give an insight into the woman behind the work of art.
Found during conservation work, the painting has been hailed as one of the most remarkable discoveries of recent times and may reveal a truer image of what the sitter looked like during the art's creation.
Art Newspaper, which published the findings, said: 'This sensational find will transform our understanding of the world's most famous picture.'
It was believed that the Prado painting was just a replica of Leonardo's cult piece and that it was made after his death.
But now it is thought that one of the artist's pupils painted it while working alongside him.
The original is on display behind glass in the Louvre, and is obscured by dark, cracked varnish, which adds years on to the woman in the picture.
Experts think it is too risky to restore or clean the painting because of its fragility.
However, the Prado version is currently being stripped of its over-paint, which art historians believe will reveal a true likeness of the model.
'It gives a much more vivid impression of her enticing eyes and enigmatic smile,' Art Newspaper said.
Experts at the Prado and the Louvre have accepted the copy's discovery, and the fact it was painted and developed alongside the master himself.
Lisa Gherardini is thought to be the woman in the painting. She was the wife of Francesco del Giocondo - a cloth merchant from Florence.
The portrait is believed to have been painted between 1503 and 1506.
Prado technical expert, Ana Gonzalez Mozo, presented the discoveries in January at a Leonardo da Vinci conference at London's National Gallery.
She said the most exciting aspect of the Prado replica is what it uncovers about Leonardo's version.
Some parts of the Madrid version have been preserved to a better standard than in the Louvre original.
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