Mary Stuart, best known as Mary Queen of Scots, was the Catholic heiress to Scotland's throne. While not mentioned in Henry VIII's succession will, the strikingly beautiful princess was related to the Tudor line and had some claim to the throne. Although most English Catholics recognized Elizabeth's rule, the Catholic world officially denied the legality of Henry VIII's marriage to Ann Boleyn, since they did not recognize the annulment of his marriage to Catherine of Aragon. Under the Catholic interpretation, this made Elizabeth illegitimate and unfit to rule. If Elizabeth was not correctly qualified by lineage to rule England, Mary Queen of Scots, conveniently a Catholic, had one of the strongest claims. Catholics throughout Europe, including some in England, believed that Mary was the true heir to the English crown. In 1580, Pope Gregory XIII announced that killing Elizabeth would not count as a sin--nor would the murder of William the Silent, a leader of the Dutch resistance against the Spanish. When William was indeed killed in 1584, Elizabeth and her advisors became increasingly fearful.
Mary Queen of Scots was married to Francis II, the King of France, who died in 1561. At this time, the widowed Queen returned to Scotland with vague hopes of taking the English throne by a coup. In 1567, Mary's subjects assassinated her husband Darnley. Following that, she married another unpopular man named Bothwell. Soon, in 1568, the Scots drove Mary out of power. Elizabeth, along with all of the rest of Europe's rulers, was horrified at the idea that the common people might revolt against their ruler. Elizabeth, fearing that Mary might go abroad and raise an army, and also afraid that the people of Scotland might lock her up, acted quickly to imprison her nemesis in Lochleven Castle, from which Mary successfully plotted her escape. Several plots against Elizabeth were discovered in the following years: the Ridolfi Plot (1571), the Duke de Guise Plot (1582), and the Babington Plot (1586). All of these failed plots hoped to assassinate Elizabeth and wanted the Spanish Army, which Philip II had then sent to suppress Protestants in the Netherlands, to invade England.
In 1584, Parliament required all English men to sign the Bond of Association, by which they promised to help hunt down anyone who killed Elizabeth. In 1585, Parliament then passed the Act for the Preservation of the Queen's Safety. In October of 1586, Mary Queen of Scots was found guilty of complicity in the Babington Plot to overthrow the Queen. Elizabeth signed Mary's death warrant, and Walsingham and Burleigh rushed her execution through without waiting to hear more from Elizabeth. On February 8, 1587, stoically facing her death, Mary Queen of Scots was executed.
Even before Mary Queen of Scots had become a threat, her mother presented some problems for Elizabeth: soon after Elizabeth's coronation, Mary of Guise, Queen Regent and the mother of Mary Queen of Scots, found herself facing opposition from Protestant reformers in Scotland called the "Lords of the Congregation." The Protestant group requested Elizabeth's aid. Elizabeth certainly wanted to get Mary of Guise's French armies out of Scotland, but she was afraid that her interference might provoke a French invasion of England. Elizabeth refused to send troops, instead sending money to support the Protestants, who overthrew the Queen Regent. Lord Burleigh (then William Cecil) encouraged military, while Elizabeth, fearful of action as always, hesitated. Ultimately, Elizabeth used the Navy to cut off French supplies to Mary of Guise's forces in Scotland. Later, at Cecil's advice, she sent an army, which was defeated. After Mary of Guise became ill, Cecil negotiated the Treaty of Edinburgh, a favorable treaty to Elizabeth. It prevented Mary of Guise and her family from using England's coat of Arms, it forced them to formally recognize Elizabeth as Queen of England, and it reacquired the removal of French forces from Scotland. Thus Elizabeth had a long history of conflict with Mary Queen of Scots' family. Mary Stuart herself refused to ratify the Treaty of Edinburgh.
Other than her later battles against the Spanish Armada, the greatest threat Elizabeth faced during her rule was from Mary Queen of Scots. But although Catholics throughout Europe supported her cause, Mary was eventually sorely disappointed in her expectation that English Catholics would rise against Elizabeth. Regardless of their religious beliefs, all of England could appreciate the welcome prosperity and stability Elizabeth's reign had provided. Furthermore, although Europe's Catholic rulers supported Mary Queen of Scots in principle, they did not actually assist her, although Elizabeth and Walsingham always feared this possibility. While it was initially impossible to link Mary to the anti-Elizabeth plots, the constant danger Mary posed motivated Parliament to push for her execution.
It was Walsingham's crafty espionage that uncovered Mary Queen of Scot's involvement in the Babington Plot. Since the execution was rushed through by Elizabeth's advisors (who claimed that they did it to spare her the pain of having to order Mary's death) the Queen later claimed that she would not actually have allowed the execution, and that her advisors had betrayed her wishes. This is unlikely, as Mary Queen of Scots was a tremendous threat. Indeed, some scholars speculate that Elizabeth herself secretly orchestrated the execution ahead of schedule, or led her advisors to think she knew they were ordering the execution without actually saying anything, so that later she would be absolved of any guilt. In the end, the question of her complicity hardly mattered, however: the people of London celebrated wildly upon Mary's death.