“Holbein painted his second miniature of Anna, this time as queen consort, at some point in early 1540. Thanks to research by Franny Moyle, whose recent biography on Hans Holbein is quite comprehensive, it is now known that a miniature sometimes identified as Katheryn Howard, Henry's fifth wife, is more likely to be of Anna. The miniature is mounted on a four-of-diamonds playing card, a nod to Anna being Henry's fourth wife. Beyond this less than subtle element, using the evidence of our eyes, one can see that Anna of Cleves is the individual in the 1540 Holbein miniature.
The sitter wears the Consort's Necklace, with its combination of ruby, diamond and pearl in the jewel standing for hope, faith and charity. The hair colour of the sitter in this miniature matches Anna's braids in the Rosenkranzbruderschaft altarpiece from 1528. There are the physical features of the sitter as well, in particular her hooded eyelids. The very same eyelids are seen in the Louvre and Rosenbach portraits and the Holbein 1539 miniature of Anna. When specifically comparing the 1539 and 1540 miniatures, the eyebrows are the same and the nose, chin and mouth bear striking similarities.
The sitter's style of dress matches contemporary descriptions of Anna in that the woman is wearing the French fashion. As mentioned above, the sitter is wearing a high-collared shirt, which was popular in German fashion in the 1540s. Katharine of Aragon wore high-collared shirts as well, perhaps showing that both women sought to instil a sense of modesty in their dress. It has been hinted at that there are even stylized escarbuncles on the sitter's green sleeve, although it is hard to tell with any certainty. The portrait shows an adult woman, and Anna was twenty-four years old when she became queen. This is very different from Katheryn Howard, who was still a teenager when she married Henry. Given the fate of Katheryn Howard, it is possible that Henry had all images of her destroyed, much as he did for Anne Boleyn. For these reasons and more, the 1540 Holbein miniature should be recognised as depicting Anna of Cleves, not Katheryn Howard.”
— Heather R. Darsie, Children of the House of Cleves