Lady Macbeth is the focus of much of the exploration of gender roles in Macbeth. As Lady Macbeth propels her husband toward committing Duncan's murder, she indicates that she must take on masculine characteristics. Her most famous speech addresses this issue. In Act I, Scene 5, after reading Macbeth's letter in which he details the witches' prophecy and informs her of Duncan's impending visit to their castle, Lady Macbeth indicates her desire to lose her feminine qualities and gain masculine ones. She cries, "Come, you spirits / That tend on mortal thoughts! unsex me here, / And fill me from the crown to the toe top full / Of direst cruelty" (I.5.38-41).

This request is part of what David Bevington, in his introduction to Macbeth in the fourth edition of the Complete Works of Shakespeare, sees as "sexual inversion" in the play. Clearly, gender is out of its traditional order. This disruption of gender roles is also presented through Lady Macbeth's usurpation of the dominant role in the Macbeth's marriage; on many occasions, she rules her husband and dictates his actions.

The disruption of gender roles is also represented in the weird sisters. Their very status as witches is a violation of how women were expected to behave in Early Modern England. The trio is perceived as violating nature, and despite their designation as sisters, the gender of these characters is also ambiguous. Upon encountering them, Banquo says, "You should be women, / And yet your beards forbid me to interpret / That you are so" (I.2.45-47). Their facial hair symbolizes their influence in the affairs of the male-dominated warrior society of Scotland. William C. Carroll, in his Bedford Cultural edition of Macbeth, sees the witches and the question of their gender as a device Shakespeare uses to criticizes the male-dominated culture, where titles are acquired through what Carroll describes as "murderous violence."