Wroth's famous work The Countesse of Montgomeries Urania was published in 1621 and introduced controversial themes concerning gender. Mary Wroth was a radical in her time merely for writing a work intended for public consumption. For the time, the act of composing a novel by a woman violated the ideals of female virtue. Bernadette Andrea, a literary critic who focuses on gender themes in Urania in her work "Pamphilia's Cabinet: Gendered Authorship and Empire in Lady Mary Wroth's Urania", writes that female virtues at the time were seen to be silence and obedience. In genteel society, an unmarried woman of the time was expected to be chaste, silent, and obedient and this theme is reiterated throughout contemporary religious works, legal treaties, and literature. The three themes were considered linked: a woman's silence and obedience were considered evidence of her chastity. By writing a text intended for a public audience, critics such as Bernadette Andrea claim that Wroth was acting against the accepted ideals of the established patriarchy and so calling her own moral character into question.
Urania is the titular character of the work, but is not the character that represents Wroth in the work. In the work, Urania is an orphan and is the daughter of a shepherd. She is actually the biological offspring of the daughter of the King of Naples, and comes to this realization over the course of the work through a series of pastoral songs and sonnets with the shepherds. The female character of Pamphilia reflects Wroth the most and is the character who struggles with the mindset of the contemporary world in which Wroth wrote. Pamphila, which is Greek for all loving, struggles throughout the text with the infidelity of her lover Amphialanthus, which is Greek for "one with two loves." Pamphilia must conceal her songs so that her moral character is not called into question by others in Urania. Pamphila carries around her works in a little cabinet and keeps them to herself because society would shun them. She is however, rewarded in the work for her actions. She becomes a queen in Asia Minor despite her lack of the contemporary virtues of silence, chastity, and obedience. Her compositions, although mostly secret, are still a violation of the code of what a woman should be and Wroth does not demonize her transgression, but rather glorifies it.
Wroth did not fare as well as her fictional character did when Urania was published. Wroth also angered people by drawing upon her contemporaries as inspiration. Paul Salzman, in The Review of English Studies article, "Contemporary References in Wroth’s Urania" notes that this work was full of references to others. One of her contemporaries claims that the "whole world condemns" her work. Denny recommends that Wroth would be better served to do as her aunt before her had done and confine herself to translating holy works and read the biblical psalms like good women of the time were expected to do. Wroth’s use of contemporaries as inspiration throughout the book has not been exactly noted. What is known is that society caught on to them and rejected the book out of hand as shameful gossip by a sinful woman who was sinning by writing a book containing her thoughts. In the article "'Not much to be marked': Narrative of the Woman's Part in Lady Mary Wroth's Urania" by Naomi Miller published in the journal Studies in English Literature, the author relates that Wroth’s novel was the first work of fiction written by an English woman to be published in the Renaissance. Virginia Woolf correctly claimed that any woman who composed a work of fiction during the period of the Renaissance would be "thought a monster". The social backlash against her work caused Urania to be pulled from publication six months after it was first produced. One critic, Lord Denny, called Wroth a "hermaphrodite in show, indeed a monster" because of the attacks he perceived Wroth to be leveling at English society and the English Court of King James in particular. Denny goes on to command, drawing on the perceived virtue of female obedience, that Wroth "leave idle books alone for wiser and worthier women have written none." She was spurned by society of the time and has only recently moved beyond being viewed as victim to being viewed as a capable author who has captured the mind of a female poet in a time where such a profession was viewed as an aberration.