Photo: archive of Prague Castle Administration

 The origins of the Prague Castle Picture Gallery date back to the turn of the 16th and 17th centuries, during the reign of Rudolf II, Holy Roman Emperor, King of Bohemia, and Archduke of Austria. From 1583, when Rudolf II asked the Bohemian Estates for a contribution toward the embellishment of Prague Castle, not only were paintings and sculptures purchased throughout Europe and artworks commissioned for the collection, but several halls were also built for their display. The space chosen was above the previously built Stables, between today's Second Courtyard and the Stag Moat, and also in the building separating today's Second and Third Courtyards. The largest of the halls, now known as the Spanish Hall, was completed in 1606. The entire collection at the time of Rudolf II's death in 1612 comprised around 3,000 works.

During the subsequent Thirty Years' War, some of the paintings were taken to Vienna. A substantial portion fell prey, just a few days before the end of the war, to a targeted raid by Swedish forces ordered by Queen Christina at the instigation of Jan Amos Comenius, who wished to protect the treasures of the Bohemian Kingdom from the Catholic Habsburgs. The Swedes had a precise description of the collection in the form of the imperial inventory of 1647. Over 500 paintings thus entered Swedish collections, where most of them remain to this day. In 1649 a new inventory was drawn up at Prague Castle, listing only two paintings and many empty frames.

Since Prague Castle was to continue serving the Habsburgs as a representative residence, some paintings were transferred back from Vienna and further works were purchased. The restored picture gallery contained approximately 600 paintings by 1656 at the latest, and although it was considerably smaller, it was in no way inferior in artistic quality to either the original collection or the Viennese collections. During the 18th century several paintings were moved to Vienna and others back to Prague, but the Gallery as such remained intact until the reconstruction of Prague Castle under the reign of Maria Theresa. The Gallery as an independent space was dissolved in 1762. The paintings no longer served as a standalone collection in a dedicated space, but merely as decoration for the royal apartments. A large group of paintings was sold to Dresden, and the remaining "surplus" paintings were sold at auction. The original spaces intended for the presentation of the Prague Castle Picture Gallery were never used for this purpose again after the Theresian reconstruction and to this day serve as representative halls.

After 1918, the newly established Czechoslovak Republic demanded the return of the removed paintings from Austria. The Austrian side acknowledged the claim but offered only inferior paintings in return, so no agreement was reached. Subsequently, the spaces of Prague Castle were redecorated with paintings purchased from the so-called Masaryk Fund for National Culture, and part of the old collections was loaned to the National Gallery. No independent picture gallery existed at the Castle. In 1961, a further group of paintings with then "unsuitable" religious subject matter was transferred to the State Institute for the Preservation of Historic Monuments. This completed the fragmentation and reduction of the original collections.

The changing atmosphere in Czechoslovakia in the 1960s, however, contributed positively to the fact that after long efforts, in 1964–1965, the original stables beneath the Spanish Hall were converted and a new Prague Castle Picture Gallery came into being. Under the direction of Professor Jaromír Neumann, by decision of the then President Antonín Novotný, the original intention of Emperor Rudolf II was revived, and the exhibition — though numerically only a fraction of the original collections — returned to Prague Castle, notable for the quality of its individual works of art. For the first time it became freely accessible to the public and was moreover placed in the context of other European galleries; some of its unique works continue to be loaned to exhibitions both at home and abroad.

After 1989, Prague Castle began searching for a new identity: many spaces that had previously been closed were opened to visitors, and much was reconstructed and changed. The effort to give the Castle a newer, more modern appearance also led to the decision to transform the Prague Castle Picture Gallery. Between 1995 and 1998, all spaces underwent a major technical reconstruction in accordance with modern security and climate-control standards. Further modifications to the exhibition spaces, a new entrance design, and interior seating were carried out according to a project by Bořek Šípek, invited to the Castle by President Václav Havel. Several paintings from the original Rudolf collection were also successfully acquired. Only the most significant pieces of historic and modern painting were selected for the permanent exhibition. Within the Gallery, individual works were arranged according to their respective local schools of painting. The works of Rudolphine masters and pieces from the collections of Emperor Rudolf II also have their own dedicated space. The author of the most recent concept of the Prague Castle Picture Gallery is PhDr. Eliška Fučíková, who had already been Professor Neumann's assistant in the 1960s and devoted herself to research into the history of the Prague Castle collections, including the comparison of individual historical inventories and other documents that make it possible to clarify the development of these collections from the 16th century to the present day. The collection of this new Gallery received international recognition in 2001, when it was exhibited as a whole in Maastricht.