When this painting of Queen Elizabeth I was last displayed to the country in 1921, curators at the National Portrait Gallery noticed spots of discolouration which cast a spiralling shadow across the Tudor posy the monarch held in her right hand. The gallery put the discrepancy down to wear and tear, and removed the work – created by an unknown artist in the 1580s or early 1590s – from permanent display.

Ironically, it is that very deterioration which has now led specialists to make a startling discovery: the anonymous artist who painted the Virgin Queen had originally depicted her clasping a snake, coiled suggestively around her right hand.

However, the artist appeared to have panicked at the last minute about depicting the Queen holding a serpent – associated with evil and original sin in Christian iconography – and hastily replaced it with an anodyne image of Tudor roses.

Tarnya Cooper, curator of the National Portrait Gallery's 16th-century collection, said she thought the painter had probably become fearful that the image would prove too shocking to a prudish British public.

Dr Cooper pointed out that the designs for Elizabeth's many portraits in her lifetime were tightly controlled by the spin doctors of her court, and that the artist may have thought his invention would not be to their liking. The snake was painted with some care, with blue and green scales.

As the centuries wore away the top layer of paint on the canvas, curators became steadily more convinced that something else lay beneath the roses which Elizabeth is holding. It was not until the work was analysed with X-ray and infra-red technology two months ago that the snake's shape was discovered. "What we now believe is that the serpent was completely finished before it was quickly painted over at the very final stages, just before the final varnish was applied," said Dr Cooper.

The Queen was known to have some items of jewellery in a snake-like form, Dr Cooper added, but an image of the serpent wrapping itself so seductively around the Queen's hand may well have caused outrage. "Maybe the serpent was too difficult and ambiguous a symbol, maybe it was too dangerous an emblem. The fact that it was painted out so quickly suggests it was too difficult a symbol for the public to cope with," she said.

David Starkey, the historian and Tudor specialist, said serpents in the 16th century held many meanings. "There was an enormous range of symbolism in the Elizabethan period and the serpent had a dual symbolism... it was undoubtedly a symbol of wisdom. There is at least one painting that shows Elizabeth I with images of green serpents on an orange taffeta dress."

The painting will be displayed at the National Portrait Gallery, for the first time since 1921, as the highlight in a four-painting display entitled Concealed and Revealed: The Changing Faces of Elizabeth I, from 13 March. It was donated to the National Portrait Gallery by a mining company, MINES Royal, the Mineral and Battery Society, in 1865.