One of the themes is the exploration of the main characters' respective identities. Gertrude Stein was inspired by the works of psychologist William James (considered to be the father of American psychology). James pioneered or helped progress the psychological-philosophical concept of pragmatism, and elements of this are evident in Three Lives. Stein articulates how the three women—Anna, Lena and Melanctha—try to express their identities by exploring their consciousness.

Within the theme of trying to express one's identity—as a woman in particular and as a human being in general—we see the complicated social and emotional dynamics between men and women, especially when they enter marriage. This is depicted by Lena's story, in which her character is seen as someone who has no will of her own. Lena represents the voiceless, or those who choose to conform to society's roles for women in the household.

Another theme of the book is the socioeconomic gaps in America and the struggle of lower-class women. Other depictions of female struggles are also present, such as Melanctha's abuse by her father as a teenager. In addition, Melanctha's sexual liberation contradicts society's oppression or criticism of such attitudes from women, especially black women, like Melanctha.

In this regard, feminism can also be considered as a theme of the book, and manifestos of female empowerment can be seen in the story, such as Melanctha's declaration after her abuse that she must use her "power as a woman" to gain a position in a male-dominated society.