Contrary to the play and the film, Mary and Elizabeth never met.

According to A. Scott Berg's memoir "Kate Remembered", Katharine Hepburn was already chosen for Mary, but they had trouble casting Elizabeth. At one point Hepburn, who had by then been nicknamed "Katharine of Arrogance", suggested that she play both roles. Supporting player John Carradine asked, "But if you played both queens, how would you know which one to upstage?" She was not amused at the time, but roared with laughter when retelling the story years later.

Katharine Hepburn, who played Queen Mary, is actually a distant relative of the Earl of Bothwell, whose family name was, in fact, Hepburn.

Katharine Hepburn credited John Ford with saving her life one day on the set. They were shooting a scene of Hepburn on horseback when the horse she was riding kept going unexpectedly. Ford yelled at Hepburn to duck just before she was about to collide with a low branch.

John Ford lost interest in this film early on. He didn't think the story was very strong, and didn't like the blank verse dialog. The film did not do well at the box office and Ford seldom mentioned it in conversation. Later, during filming of Stagecoach (1939), Ford harassed several actors, notably John Wayne, about their performances. As he began with Thomas Mitchell, who played Doc Boone, Mitchell reportedly said, "Just remember, I saw 'Mary of Scotland'". Ford left him alone for the remainder of the shoot.

Ginger Rogers, posing as British actress "Lady Ainsley" in hopes of landing the role of Queen Elizabeth, tested with an unsuspecting 'Katherine Hepburn'. During the test, Hepburn who also wanted the role, became aware of the lavish subterfuge created by Mel Berns, RKO's head of makeup, who with Leland Hayward, plotted to dupe director John Ford into offering the coveted role to Rogers. Archived film of the silent test caught Hepburn kicking Rogers in the shins. Instead, the role went to Florence Eldridge. Hepburn got even with Rogers by pouring water on her new fur coat saying "If it is real mink, it won't shrink."

According to Katharine Hepburn, during the filming of Mary and Bothwell's love scene, John Ford, rather fed up with the idea of directing a romantic costume drama written in blank verse, simply said to Hepburn, "Here; you direct this scene." And she did.

Both Ginger Rogers and Bette Davis were interested in playing Elizabeth. Director John Ford wanted Tallulah Bankhead for the part, but Florence Eldridge. Fredric March's real-life wife, won the part

Katharine Hepburn wanted George Cukor as director, but after the failure of Sylvia Scarlett (1935), producer Pandro S. Berman refused to let them work together again.

According to Katharine Hepburn's autobiography "Me", director John Ford lost interest in the film when he discovered that the plot was not particularly strong. She recalls one day, Ford announced that he was leaving early and would allow Hepburn to direct a scene with Fredric March. Hepburn feared that March would not listen to direction from her, but when he acquiesced, she directed her first and only scene.

The play opened in New York City, New York, USA on 27 November 1933 and had 248 performances. The title role was played by Helen Hayes and the cast also included Moroni Olsen, who repeated his role as John Knox in the 1936 film version, Edgar Barrier (Lord Douglas), Ernest Cossart (Lord Throckmorton) and George Coulouris (Lord Burghley). The play was written in blank verse.

David Rizzio was described by contemporaries as a dwarf. He is played by John Carradine who was six feet tall.

Leslie Goodwins filled in as director for a few scenes when John Ford wasn't available.

According to Hepburn biographer Alvin H. Marill, the actress turned down an offer from Max Reinhardt to play Viola in "Twelfth Night" at the Hollywood Bowl in order to meet her obligation for "Mary of Scotland".

Ginger Rogers was tested for the role of Queen Elizabeth I.

Moroni Olsen was the only member of the original Broadway cast of the play to repeat his role in the film version.

John Carradine completed his role as 'David Rizzio' on March 21 1936.

E.E. Clive is listed in casting records for the role of Burghley, but that role was played by Lionel Pape