Spanning the period between the American Civil War and World War I, East of Eden presents two generations of brothers as they battle between good and evil. The first generation consists of the placid Adam Trask and his hot-tempered brother Charles, and the second generation is made up of Adam's sons, the fair-haired, mild-mannered Aron and the dark-haired, quick-tempered Cal. Deeply influenced by the biblical story of Cain and Abel, the novel begins in California's Salinas Valley, where the young narrator was born. He recounts the story of his Irish grandparents, Samuel and Liza Hamilton, who settle in the Salinas Valley, where they meet the members of the Trask family.
The first part of the novel concerns the first generation of Trask brothers, Adam and Charles, and Adam's wife, Cathy Ames (who is referred to as "Kate" in the second part of the book). Cyrus Trask, the one-legged, crooked father, commits the "original sin" that inspires the action of the novel. Cyrus loves his son Adam more than his other son, Charles, and favors Adam's gift of a puppy over Charles's gift of an expensive knife. Tormented by jealousy, Charles savagely beats his brother. Cyrus also lies about his Civil War record to win an important job in Washington D.C. Through his crooked financial dealings, he is able to leave his sons an inheritance of $100,000, which Adam finds out about when he returns from the army. Meanwhile, the most evil character in the novel, Cathy Ames, murders her parents by setting fire to their home. She then becomes the mistress of Mr. Edwards, who runs a ring of prostitutes. Adam marries Cathy soon after she wanders unto his farm, near-dead from a beating suffered at the hands of Edwards. Together, they move to Salinas, where they employ the Chinese-American Lee as a cook and housekeeper, and get to know the much-beloved Samuel Hamilton, who mentors and guides Adam. In Salinas, Cathy gives birth to twin sons, Aron and Cal, and almost immediately abandons her family to return to prostitution, but only after shooting Adam when he attempts to stop her. Soon afterwards, she poisons Faye, the brothel-owner, in an attempt to take over the business. She gives her whores drugs, encourages sadomasochistic sexual practices, and blackmails her customers.
The next part of the novel centers on Adam's children, Aron and Cal, and on Abra Bacon, who falls in love with both brothers. At heart a scholar, Lee raises the twins and remains a constant friend to his employer, Adam Trask, and to their neighbor, Samuel Hamilton, who is raising nine children himself. After a scholarly discussion with Adam and Samuel, Lee researches the Cain and Abel biblical story and proffers the novel's central concept of timshel ("thou mayest"). As the twins grow older, Aron and Cal vie for their father's affection, much as Adam and Charles did earlier. Cal is by far the more complex brother, and knows full well that his father loves Aron more because he resembles their mother. As a teenager, Cal gambles, visits brothels, and is consumed by jealousy over Abra Bacon's love for Aron. Meanwhile, Aron falls in love with Abra, who returns his childish love but realizes later on that Aron, who plans to enter the ministry, is only in love with her glorified image. The daughter of a crooked Salinas politician, Abra comes to love Cal instead, and like him, wonders whether a parent's evil nature can be inherited. In an attempt to gain his father's love, Cal decides to go into business to raise $15,000 to recover his father Adam's business losses. The monetary gift, however, results in disaster: Adam reacts violently when he learns that Cal took advantage of farmers during war-time in an effort to make the money. However, like his father before him, Adam fails to see the love behind his son's gift. In a jealous rage, Cal decides to take Aron to their mother's brothel. All the while, Aron had believed his mother to be dead, and when he finds out otherwise, he runs away, joins the Army during World War I, and dies soon after. After meeting Aron, Cathy commits suicide.
Ultimately, East of Eden deals with themes of intergenerational sin, consequent guilt, redemption and forgiveness. Adam suffers a stroke after hearing of Aron's death, and Cal feels overwhelmed with guilt. The negative family legacy is stopped dead in its tracks, however, when Cal is offered surcease from his guilt. His father Adam lifts his hand in a final blessing and utters the word "timshel", the Hebrew word for "thou mayest". Finally, Cal realizes that he is not predestined to live a life of evil, but has the free will to choose goodness and morality.