A measure of the importance of Holyroodhouse is the status of its Keeper, who was appointed to oversee the Palace in the absence of the court. There were various grants of the office of Keeper of Holyroodhouse until 1646 when Charles I conferred it heritably on the 1st Duke of Hamilton, whose descendants have retained it ever since. The post is one of the Great Offices in the Royal Household in Scotland. As well as his own deputy, the Keeper still appoints the Bailie of Holyroodhouse, who is responsible for law and order within the Holyrood Abbey Sanctuary.

There was formerly a separate hereditary Keeper of Holyrood Park, which surrounds Holyroodhouse, held by the Earls of Haddington. This was purchased by the Crown and the office extinguished in 1843 after disputes over the Keeper's right to allow quarrying within the Park.