Book I is centered on the virtue of Holiness as embodied in the Redcrosse Knight. He and his lady Una travel together as he fights the dragon Errour, then separate as the wizard Archimago tricks the Redcrosse Knight in a dream to think that Una is unchaste. After he leaves, the Redcrosse Knight meets Duessa, who feigns distress in order to entrap him. Duessa leads the Redcrosse Knight to captivity by the giant Orgoglio. Meanwhile, Una overcomes peril, meets Arthur, and finally finds the Redcrosse Knight and rescues him from his capture, from Duessa, and from Despair. Una and Arthur help the Redcrosse Knight recover in the House of Holiness, with the House's ruler Caelia and her three daughters joining them; there the Redcrosse Knight sees a vision of his future. He then returns Una to her parents' castle and rescues them from a dragon, and the two are betrothed after resisting Archimago one last time.
Book II is centred on the virtue of Temperance as embodied in Sir Guyon, who is tempted by the fleeing Archimago into nearly attacking the Redcrosse Knight. Guyon discovers a woman killing herself out of grief for having her lover tempted and bewitched by the witch Acrasia and killed. Guyon swears a vow to avenge them and protect their child. Guyon on his quest starts and stops fighting several evil, rash, or tricked knights and meets Arthur. Finally, they come to Acrasia's Island and the Bower of Bliss, where Guyon resists temptations to violence, idleness, and lust. Guyon captures Acrasia in a net, destroys the Bower, and rescues those imprisoned there.
Book III is centred on the virtue of Chastity as embodied in Britomart, a lady knight. Resting after the events of Book II, Guyon and Arthur meet Britomart, who wins a joust with Guyon. They separate as Arthur and Guyon leave to rescue Florimell, while Britomart rescues the Redcrosse Knight. Britomart reveals to the Redcrosse Knight that she is pursuing Sir Artegall because she is destined to marry him. The Redcrosse Knight defends Artegall and they meet Merlin, who explains more carefully Britomart's destiny to found the English monarchy. Britomart leaves and fights Sir Marinell. Arthur looks for Florimell, joined later by Sir Satyrane and Britomart, and they witness and resist sexual temptation. Britomart separates from them and meets Sir Scudamore, looking for his captured lady Amoret. Britomart alone is able to rescue Amoret from the wizard Busirane. Unfortunately, when they emerge from the castle Scudamore is gone. (The 1590 version with Books I–III depicts the lovers' happy reunion, but this was changed in the 1596 version which contained all six books.)
Book IV, despite its title "The Legend of Cambell and Telamond or Of Friendship", Cambell's companion in Book IV is actually named Triamond, and the plot does not center on their friendship; the two men appear only briefly in the story. The book is largely a continuation of events begun in Book III. First, Scudamore is convinced by the hag Ate (discord) that Britomart has run off with Amoret and becomes jealous. A three-day tournament is then held by Satyrane, where Britomart beats Arthegal (both in disguise). Scudamore and Arthegal unite against Britomart, but when her helmet comes off in battle Arthegal falls in love with her. He surrenders, removes his helmet, and Britomart recognizes him as the man in the enchanted mirror. Arthegal pledges his love to her but must first leave and complete his quest. Scudamore, upon discovering Britomart's gender, realizes his mistake and asks after his lady, but by this time Britomart has lost Amoret, and she and Scudamore embark together on a search for her. The reader discovers that Amoret was abducted by a savage man and is imprisoned in his cave. One day Amoret darts out past the savage and is rescued from him by the squire Timias and Belphoebe. Arthur then appears, offering his service as a knight to the lost woman. She accepts, and after a couple of trials on the way, Arthur and Amoret finally happen across Scudamore and Britomart. The two lovers are reunited. Wrapping up a different plotline from Book III, the recently recovered Marinel discovers Florimell suffering in Proteus' dungeon. He returns home and becomes sick with love and pity. Eventually he confesses his feelings to his mother, and she pleads with Neptune to have the girl released, which the god grants.
Book V is centred on the virtue of Justice as embodied in Sir Artegall.
Book VI is centred on the virtue of Courtesy as embodied in Sir Calidore.