Portraits of Henry VIII, his third wife Jane Seymour and their son Edward are to be hung together for the first time since Henry himself owned them.
The Henry VIII portrait is thought to date from 1537
The works by 16th Century court painter Hans Holbein the Younger will go on display in an exhibition at the Tate Britain in London in September.
But the German artist's masterpiece The Ambassadors will not be on show.
The National Gallery has refused to lend the "wafer thin" work, fearing the move might split its wooden panel.
The painting was restored by the National Gallery in the late 1990s.
The exhibition at Tate Britain, titled Holbein in England, will run until 7 January.
It will feature 30 portrait drawings, six paintings and several miniatures from the Royal Collection, along with some of Holbein's jewellery designs.
"Holbein survived when others were losing their heads," the exhibition's curator, Dr Susan Foister said. "He opened the door to a wave of portraiture."
Jane Seymour gave Henry VIII a much wanted male heir
The oil panel portrait of Henry VIII, dating from 1537, is being loaned from the Thyssen-Bornemisza Museum in Madrid, while the Seymour portrait is being borrowed from a museum in Vienna.
Holbein's portrait of Edward, Prince of Wales, is on loan from the National Gallery of Arts in Washington.
The artist, whose father was also a painter, was born in Germany in 1497 or 1498 and moved to England when he was in his late twenties.
Dr Foister said: "He was a survivor. After the break with Rome, when England was set on the path to Protestant reformation, he launched a line in Protestant imagery."
The exhibition will be the artist's first major UK show since Holbein and Other Masters was staged at the Royal Academy in 1950.
Holbein is thought to have been buried in either the Church of St Andrew Undershaft or the Church of St Katherine Cree, both in Leadenhall Street, London.