The beginning of the Prague Castle Picture Gallery goes back to the late 16th and early 17th century, the period of the rule of Rudolf II – Holy Roman Emperor, King of Bohemia, and Archduke of Austria. After 1583, when Rudolf II asked the Bohemian estates for their contribution to the expansion of Prague Castle, not only paintings and statues from all over Europe were purchased and artworks were commissioned specifically for this collection, but also several halls for their display were completed. The space was designated above the previously erected stables between the present-day Second Courtyard and Stag Moat and also in the building that currently separates the Second and Third Courtyards. The largest hall, today called Spanish Hall, was completed in 1606. The entire collection comprised about 3,000 works of art in 1612 when Rudolf II died.

During the Thirty Years’ War that followed, a portion of the paintings was transferred to Vienna. Several days before the war ended, a substantial number became war spoils for the invading Swedish army, commanded by Queen Christina at the behest of John Amos Comenius in support of his efforts to save the treasures of the Kingdom of Bohemia from the Catholic Habsburgs. The Swedes had a concrete description of the collection – the complete imperial inventory list from 1647. Over 500 paintings thus arrived in the Swedish collection and most of them currently remain there. In 1649, a new inventory list was compiled at Prague Castle that consisted of only two paintings and many empty frames.

Because Prague Castle ultimately served as the Habsburgs’ representative residence, a portion of the paintings returned from Vienna and additional works were purchased. No later than 1656, the renewed picture gallery consisted of about 600 paintings, and although it was substantially smaller, its artistic value did not lag behind the original collection or the Viennese collections. During the 18th century, several paintings were transferred to Vienna and others to Prague but the Picture Gallery remained intact until the renovation of Prague Castle under Maria Theresa. As an individual space, the Picture Gallery folded in 1762. The paintings no longer constituted a collection in a gallery but rather served as a mere decoration of the royal apartments. A large set of paintings was sold to Dresden, while the remaining ‘excessive’ paintings were sold at an auction. After the renovation under Maria Theresa, the original space of the Prague Castle Picture Gallery no longer served its purpose and it has been used for representative rooms ever since.

After 1918, the newly established Czechoslovak Republic requested the return of paintings from Austria. Even though the Austrian side recognised this claim, it offered to return only second-rate paintings; therefore, the agreement failed. Subsequently, the Prague Castle rooms were decorated by paintings purchased from the so-called Masaryk National Culture Fund and part of the old collection was lent to the National Gallery. There was no self-contained picture gallery at Prague Castle. In 1961, another portion of paintings with the ‘inapt’ religious themes was handed over to the State Institute of Heritage Care, which finalised the dissipation and reduction of the original collection.

Nevertheless, following long efforts, the changing atmosphere in Czechoslovakia during the 1960s contributed to the reconstruction of the original stables under the Spanish Hall and the origin of a new Prague Castle Picture Gallery in the years 1964–1965. Spearheaded by Professor Jaromír Neumann and based on the resolution of then President Antonín Novotný, the original intention of Emperor Rudolf II was fulfilled and the gallery returned to Prague Castle; although it contains only a fragment of the original collection, it includes works of art of exceptional quality. It opened to the public for the first time and, moreover, reached the quality level of other European galleries; some unique works are still being lent to exhibitions at home and abroad.

After 1989, Prague Castle sought a new character, opening to visitors many spaces that were previously closed, and reconstructing and changing many areas. The effort to impart to Prague Castle a new, modern appearance resulted in the decision to transform the Prague Castle Picture Gallery. During 1995–1998, the space underwent crucial technical reconstruction to comply with modern standards of security and indoor climate conditions. Furthermore, President Václav Havel invited Bořek Šípek to Prague Castle who redesigned the exhibition areas and the entrance, and equipped the interiors with various types of furniture for sitting areas. In addition, several paintings from Rudolf’s original collection were purchased. Only the most significant historical and modern paintings were chosen for the permanent exhibition. The works were arranged in the Prague Castle Picture Gallery according to their painting school affiliation. The paintings of Rudolf II’s masters and the works from his collection received their own space. The art historian Eliška Fučíková devised the latest concept of the Prague Castle Picture Gallery. She was an assistant to Professor Neumann in the 1960s and she participated in research of the history of Prague Castle Collections including the comparison of historical inventories and other documents that enable to clarify the development of these collections from the 16th century to the present. The new Prague Castle Picture Gallery collection received an international award in 2001 when it was displayed in its entirety in Maastricht. In 2020, a portion of the paintings was displayed at the representative exhibition The Masterpieces of the Prague Castle Picture Gallery in the National Gallery of Slovenia in Ljubljana.