Now, an Italian art historian has claimed that the model in the world's most famous painting is in fact a male and not a female, reports the Daily Mail.
According to Silvano Vinceti, the Renaissance artist's male apprentice and possible lover Gian Giacomo Caprotti was the main inspiration for the masterpiece.
Caprotti, also known as Salai, was one of his male muses, whose nose and mouth bear striking similarities to those of Mona Lisa.
He worked as an apprentice with the artist for more than two decades from 1490 and they were rumoured to have been lovers.
Some experts had already suggested that Vinci could have based his masterpiece on a self-portrait.
But Vinceti, a researcher who has been analysing the painting using state-of-the-art high-magnification techniques, also claimed to have found the letter 'S' in the model's eyes, which may be a reference to Salai.
Several of Leonardo's works, including St John the Baptist and a drawing called Angel Incarnate, are said to have been based on Salai.
Vinceti, president of Italy's National Committee for Cultural Heritage, said these paintings depict a slender, effeminate young man with long auburn curls and almost identical facial features to the Mona Lisa.
"Salai was a favourite model for Leonardo. Leonardo certainly inserted characteristics of Salai in the last version of the Mona Lisa," he said.
Most experts believe the model for the Mona Lisa, which hangs at the Louvre in Paris, was Lisa Gherardini, the 24-year-old wife of a rich Florentine silk merchant.
They say Leonardo started painting her in 1503. But Vinceti claims he may have started in the late 1490s in Milan, coinciding with the time he built up a relationship with Salai.
His claims have caused a stir in the art world, with many dismissing the idea that Mona Lisa was a man.
Da Vinci expert Pietro Marani said the theory was 'groundless'.
"All Leonardo subjects look like each other because he represents an abstract ideal of beauty," said the art professor at Milan's Politecnico University.
"They all have this dual characteristic of masculine and feminine," he added.
Image: The Mona Lisa by Leonardo da Vinci | Photograph: Wikimedia
Decoding Da Vinci's Mona Lisa
Doctor with an eye for style
Photo: Ash's Mona Lisa look
Himesh woos Mona Lisa