Based on Maxwell Anderson's play, the Scot queen chooses a husband, regrets it, marries for love, faces civil war, flees, is imprisoned and executed.
England's Queen Elizabeth (Florence Eldridge) orders Mary seized at sea on her way from France to Scotland; but Mary (Katharine Hepburn) lands, prays, and goes to Holyrood castle, where James Stuart Moray (Ian Keith) welcomes his sister. Mary says she will rule justly and keeps David Rizzio (John Carradine) as her secretary. Moray was converted by John Knox, but Mary holds to her Catholic religion. Moray suggests she marry Darnley, but she refuses to marry at all. Huntly (Donald Crisp) defends her, and people outside sing for her. John Knox (Moroni Olsen) preaches against Mary; but the Earl of Bothwell (Fredric March) brings in bagpipes. Mary invites Knox inside and asks him to respect her religion; but he walks off. Mary asks Bothwell to be her friend, and he tells the council he now heads the army. The English ambassador Randolph (Ralph Forbes) reports to Elizabeth about Mary. Elizabeth sends Throckmorton (Alan Mowbray) to Moray.
Rizzio urges Mary to marry Darnley. Throckmorton tells Mary that Elizabeth wants her to marry Leicester; but Mary says she will marry Darnley (Douglas Walton), a foppish contrast to the manly Bothwell, who barges in and tells Mary he loves her. Yet Mary gives Darnley her hand. Now that Bothwell has left, Moray and the council complain that Mary listens to Rizzio. A drunk Darnley enters and says he is king; Mary walks out. Rizzio asks Darnley to sign papers. Men rouse Darnley's jealousy of Rizzio, who sings to Mary of love. Mary asks Rizzio to leave for his own sake; but soldiers come in and kill him. Mary blames Darnley. She is told to sign a pardon for the murderers; but she learns that Bothwell is coming. She asks Darnley to help her escape, and Bothwell gives her his horse. During the fighting Moray and council men flee.
Elizabeth learns that Mary has a son. Bothwell brings baby James a present. A paranoid Darnley threatens to deny his son and is killed in his castle. Knox preaches for vengeance. Bothwell takes Mary to his castle with Huntly. Mary kisses Bothwell and weds him, causing Huntly to break his sword. Moray besieges Holyrood castle. Bothwell tells Moray he will leave Scotland if Mary is sovereign. Moray and the lords agree, and Bothwell rides away. Mary tells Moray to remove Knox; but he refuses to obey her, asking her to abdicate. Throckmorton reports to Elizabeth that Bothwell is raising arms against Moray, who as regent crowned James. Elizabeth opposes Moray publicly but secretly helps him, sending her ring to Mary.
Mary escapes Scotland and is imprisoned in England. Bothwell raves in a cell and dies. A tribunal accuses Mary of plotting to kill Elizabeth, while Mary accuses Elizabeth of treachery. When Mary learns that Bothwell is dead, she loses hope. Elizabeth visits her the night before her execution, saying she is a queen, while Mary knows what it is to be a woman. Mary does not regret losing her kingdom for Bothwell and tells Elizabeth they could have been friends. The English queen asks Mary to renounce her claim to that throne; but Mary says that her son will rule England. In the final scene Mary prays before her execution.
This drama broadly describes this historic conflict, emphasizing Mary's romantic marriage to Bothwell that gave them only three weeks of bliss. Elizabeth is seen as a ruthless queen, who killed Mary to preserve her power.