An unexpected discovery by conservation staff, who found a portrait of Catherine of Aragon during a wander around Lambeth Palace, has resulted in a portrait of the former Queen being hung alongside her former husband, King Henry VIII, for the first time in nearly 500 years.

A portrait in a private sitting room at the palace, dated from the early 16th century and originally identified as Catherine Parr, turned out to be the temperamental King’s first wife after experts examined her facial features and costume.

The group, who had been making their royal visit for research, persuaded the Archbishop of Canterbury and the Church Commissioners to lend them the painting for further technical analysis.

Raking light showed the black over paint from the background of the work could be removed, revealing an original, damask silk-imitating green finish similar to an existing portrait of the King at the National Portrait Gallery, where the two portraits are now on public display.

The investigative process has produced a range of revelations. X-rays of Catherine’s headdress suggested over-painting had altered the look of her face, and the original Tudor finish of the frame – hidden beneath layers of later paint of gilding – combines oil gilding with bands of blue and red, described by conservators as “extremely rare”.

“Henry VIII and Catherine of Aragon were married for nearly 24 years, and during that time their portraits would have been displayed together in this fashion, as King and Queen of England,” said Dr Charlotte Bolland, who has overseen a five-year research project, Making Art in Tudor Britain, allowing portraits to be analysed with scientific techniques.

“It is wonderful to have the opportunity to display this important early portrait of Catherine of Aragon at the Gallery.”

• Henry and Catherine Reunited is on show in Room 1 of the National Portrait Gallery. Admission is free.