A small detail on the back of the miniature could be a potential clue to the nature of Wriothesley's personal relationship with Shakespeare, said Dr Goldring, who spent eight months studying the artwork.
"Miniatures were inherently private artworks that were frequently exchanged as love tokens," she said.
"This miniature is pasted onto a playing card, which is customary for the time.
"The reverse of this playing card was originally a red heart, but most unusually, the heart has been deliberately obliterated and painted over with a black arrow.
"It could, arguably, be a spade - but I think it more strongly resembles a spear, the symbol that appears in Shakespeare's coat of arms."
While it is impossible to say when the defacement on the card happened, Dr Goldring said it was "certainly done with a purpose".
The oval painting measures just two and a quarter inches in height.
Goldring added: "One tantalising interpretation might be that Shakespeare was the original recipient of the miniature but returned it to the Earl at some point.
"Perhaps around the time of Southampton's marriage in 1598 - with his personal mark firmly obscuring the heart.
"Such a scenario would help to explain why and how the miniature remained in a branch of the Southampton family for hundreds of years."