This important miniature of Mary, Queen of Scots is by the leading exponent of the new art of miniature painting at the court of Elizabeth I, Nicholas Hilliard. According to an early eighteenth-century account, Hilliard 'drew Mary Queen of Scots in WaterColours when she was but 18 years of Age, wherein he succeeded to Admiration and gain'd a general Applause' (Bainbrigg Buckeridge in de Piles 'The Art of Painting with the Lives of the ... most Eminent Painters', 1706). Tempting as it might be to see this miniature as the product of Hilliard's precocious talent c. 1560, its close relationship to a full-length portrait type known as the 'Sheffield' or 'Oudry' portrait of 1578 makes it more likely to date from this later period. Nicholas Hilliard was away in France from the autumn of 1576 to late 1578, employed in the household of François, duc d'Alençon, and it seems likely that the sittings for the present miniature were been given to Hilliard by Mary, Queen of Scots prior to his leaving for France in 1576 and while she was being held in captivity by the Earl of Shrewsbury. There are many references to miniatures of Mary, Queen of Scots being distributed by the Queen to her scattered supporters at this period, demonstrating the way in which this new portable art-form could be deployed effectively to cement political as well as personal affiliations.

A second version of the miniature is in the Victoria and Albert Museum, London (P.24-1975). The blue background in the present miniature is painted using the costly pigment ultramarine, in contrast to the background in the Victoria and Albert Museum's collection which is painted using azurite (bice). Both are by the hand of Nicholas Hilliard, although Reynolds considered the Victoria and Albert Museum version to be the prime, livelier example of the two. Both were also apparently in the Royal Collection in the Stuart period, the Victoria and Albert version having left the Royal Collection in the hands of James II when he fled to France in 1688, and the present miniature having been recorded in the inventory of Charles II. If it is to be associated with the miniature recovered by Col. Hawley at the Restoration ('A peice of Mary Queen of Scotland in A gold oval Case') then this may suggest that it had formerly been in the possession of Charles I.