The novel’s opening chapters establish the Civil War setting, delve into the sociopolitical debates surrounding the war, and illustrate the lives of slaves, particularly Uncle Daniel, Aunt Linda, Robert Johnson, and Tom Anderson. In the North Carolina marketplace, Tom and Robert meet to secretly discuss the Union army’s progress against the South. Soon after, the slaves desert their owners and join the Union army. Uncle Daniel leads a clandestine prayer meeting in the woods, the only safe location for slaves to congregate and worship.

Iola Leroy, a slave with a white complexion and blue eyes, is held captive by the abusive Master Tom. Tom Anderson arranges for the Union army to rescue Iola. Exiled from family and home, Iola becomes a nurse in the army. Tom is wounded in a skirmish with Confederate forces, and Iola’s attempts to restore his health are in vain. Dr. Gresham, a white Union hospital physician, develops affection for Iola but is disturbed by her tender care of Tom, a black man. Dr. Gresham despises miscegenation, or racial mixing, and checks his feelings for Iola when he learns that she is a "mulatta," a woman who is both black and white. Nevertheless, Dr. Gresham cannot suppress his feelings and proposes to Iola.

To explain Iola’s condition as a slave, the novel flashes back to her parents’ courtship and marriage. The time sequence is out of order to develop conflict and suspense. Iola’s father, Eugene Leroy, is a wealthy southern slave owner. When he becomes ill, Leroy’s friends abandon him. A compassionate slave, Marie, who is one-quarter black, tends to Leroy. Grateful for Marie’s care, the love-struck Leroy sends Marie to a northern school, frees her from slavery, and, despite protests from his cousin Alfred Lorraine, proposes to her. The couple marry, and Marie bears three children who are one-eighth black, but they decide to raise Harry, Iola, and Gracie as white. To protect them from prejudice and slavery, the Leroys lead a solitary life and later send their children to northern schools. Marie predicts that Lorraine, who shuns her and her children because they are mixed race, will sell them into slavery upon Leroy’s death.

The novel skips ahead several years to Iola’s childhood in a northern school. Ironically, Iola professes a pro-slavery stance. In a letter to her parents, she details a controversial incident at school—a black girl’s enrollment. The novel’s nonlinear structure creates irony and foreshadows conflict, for the reader already knows that Iola is an enslaved woman of mixed race. Upon Leroy’s death from yellow fever, Lorraine orchestrates the family’s demise, finagling legal loopholes to overtake Leroy’s property, to void Leroy and Marie’s marriage, and to nullify Marie’s freedom. Then, he nefariously sells Marie and her children as slaves. Marie discloses Iola’s true identity as a mixed-race woman. Marie and Gracie also catch yellow fever, and Gracie dies.

The plot shifts back to the present. Iola rebuffs Dr. Gresham’s proposal because she refuses to marry a man whose race has oppressed her family and the black community via slavery. Iola begins to accept her identity as black, and she resolves to locate her mother before committing to any marriage. Meanwhile, Harry remains at school in Maine, unaware of his family’s tragedy until he receives a letter from Iola. When he learns of his real identity and his family’s separation, he becomes ill. Later, Harry joins the Union army’s black regime. While he hesitates to associate himself with a race ostracized as lower class and inferior, Harry chooses to pass as black. Iola later nurses Robert, hurt in battle. She sings to soothe him, and he recognizes the song as his mother’s. Coincidentally, the two discover that they are relatives. Robert is Marie’s sister and Iola’s uncle. As the Civil War ends, the Union hospital closes. Iola briefly teaches at a school for freed slaves. Robert revisits the Johnson plantation, where he was enslaved, and discovers a thriving settlement of freed blacks, including Uncle Daniel and Aunt Linda. Harry awakens from a war injury with his mother unexplainably at his bedside. Within the context of religious events, the slaves’ families reunite. At a prayer meeting, Robert reclaims his long-lost mother, Harriet. Harry and Iola reconnect at a Methodist Conference.

While Marie and her children move to Georgia and relish their time together, Harriet and Robert travel to the North. Eventually, Iola and Marie reunite with Harriet. Robert and Iola confront discrimination in the northern housing market, and as a black woman, Iola struggles to find employment. The Leroy family associates with progressive northern thinkers, including Dr. Latimer, Dr. Gresham, and Miss Delany, who gather at a conversazione to discuss the post-war sociopolitical conditions facing the black race. The group aims to sway public opinion about blacks’ achievements, and the astute Dr. Latimer, a mixed-race man, proves his scholarly ability before Dr. Latrobe, a white southerner skeptical of blacks’ intellectual equality. Lorraine becomes a Confederate soldier and dies in the war.

The convoluted plot revolves around and questions the social implications of miscegenation. Iola rejects Dr. Gresham’s second proposal as she now fully perceives herself as a black woman who intends to marry a black man. Dr. Latimer decides to pass as black, sacrificing family fortune and the upward mobility granted to whites. Both fervent proponents for blacks’ rights, Dr. Latimer and Iola fall in love and marry. Harry is engaged to Miss Delany, a college-educated black woman and teacher. The plight of slave owner and slave has reversed. The slave owners have deteriorated. Master Gundover, Aunt Katie’s slave owner, died, and Mrs. Johnson, now poor, depends on Robert’s financial support. However, the former slaves prosper and return to North Carolina to elevate the black community. Dr. Latimer and Iola succeed in their respective careers as physician and teacher. Harry and Miss Delany operate a school. Robert purchases land, which he resells to the needy. While Uncle Daniel and Harriet retire, Marie volunteers in the community. The reunited Leroy family toils as advocates for racial empowerment and civil rights.