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Elizabeth's senior advisor, Burghley, died on 4 August 1598. His political mantle passed to his son, Robert Cecil, who soon became the leader of the government. One task he addressed was to prepare the way for a smooth succession. Since Elizabeth would never name her successor, Cecil was obliged to proceed in secret. He therefore entered into a coded negotiation with James VI of Scotland, who had a strong but unrecognised claim. Cecil coached the impatient James to humour Elizabeth and "secure the heart of the highest, to whose sex and quality nothing is so improper as either needless expostulations or over much curiosity in her own actions". The advice worked. James's tone delighted Elizabeth, who responded: "So trust I that you will not doubt but that your last letters are so acceptably taken as my thanks cannot be lacking for the same, but yield them to you in grateful sort". In historian J. E. Neale's view, Elizabeth may not have declared her wishes openly to James, but she made them known with "unmistakable if veiled phrases".

The Queen's health remained fair until the autumn of 1602, when a series of deaths among her friends plunged her into a severe depression. In February 1603, the death of Catherine Howard, Countess of Nottingham, the niece of her cousin and close friend Catherine, Lady Knollys, came as a particular blow. In March, Elizabeth fell sick and remained in a "settled and unremovable melancholy". She died on 24 March 1603 at Richmond Palace, between two and three in the morning. A few hours later, Cecil and the council set their plans in motion and proclaimed James VI of Scotland as king of England.

Elizabeth's coffin was carried downriver at night to Whitehall, on a barge lit with torches. At her funeral on 28 April, the coffin was taken to Westminster Abbey on a hearse drawn by four horses hung with black velvet. In the words of the chronicler John Stow:

Westminster was surcharged with multitudes of all sorts of people in their streets, houses, windows, leads and gutters, that came out to see the obsequy, and when they beheld her statue lying upon the coffin, there was such a general sighing, groaning and weeping as the like hath not been seen or known in the memory of man.

Elizabeth's funeral cortège, 1603, with banners of her royal ancestors

Elizabeth was interred in Westminster Abbey in a tomb she shares with her half-sister, Mary. The Latin inscription on their tomb, "Regno consortes & urna, hic obdormimus Elizabetha et Maria sorores, in spe resurrectionis", translates to "Consorts in realm and tomb, here we sleep, Elizabeth and Mary, sisters, in hope of resurrection".