Prado Museum said it did not realize its significance until a recent restoration revealed hidden layers.
The artwork features the same female figure, but had been covered over with black paint and varnish.
The painting is thought to have been created by one of Leonardo’s students alongside the 16th century original.
The Art Newspaper, which reported the discovery, said the “sensational find will transform our understanding of the world’s most famous picture”.
The original painting, which currently hangs at the Louvre in Paris, is obscured by several layers of old, cracked varnish.
However, cleaning and restoration is thought to be too risky because the painting is fragile.
The Art Newspaper said the removal of the black paint on the replica had revealed “the fine details of the delicate Tuscan landscape”, which mirrors the background of Leonardo’s masterpiece.
Martin Bailey, who reported on the discovery for the paper, said: “You see Lisa’s eyes, which are quite enticing, and her enigmatic smile. It actually makes her look much younger.”
In fact, the new painting has led experts to speculate that the woman who sat for the Renaissance Masterpiece was in her early 20s – much younger than the Louvre’s original appears to show.
But after using x-rays to analyze the original drawings underneath, conservators have concluded the work was carried out at the same time as Leonardo’s original.
Prado Museum presented its findings at a conference on Leonardo da Vinci at London’s National Gallery.
There is still some restoration to complete on the painting but, once it is finished, it will be exhibited at the Louvre in March, allowing visitors to compare the two works.
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